JP2023530766A - Bispecific antibody and use thereof - Google Patents

Abstract

SUMMARY The present invention discloses bispecific antibodies and uses thereof. The bispecific antibody comprises an antigen binding domain targeting B7-H4 and an antigen binding domain targeting 4-1BB. The bispecific antibody of the invention has one, two or three sites that bind 4-1BB and comprises a novel, fully human B7-H4 antibody; targeting H4 reduces toxicity due to 4-1BB activation by specifically binding to tumor cells; It retains the binding role of FcRn and has a long half-life. [Selection figure] None

Description

This application claims the priority of Chinese patent application 202010618149.1 with filing date 2020/6/30. The present application cites the full text of the aforementioned Chinese patent application.

The present invention relates to the field of biopharmaceuticals, and in particular to bispecific antibodies, especially bispecific antibodies targeting B7-H4 and 4-1BB, and uses thereof.

B7-H4 (VTCN1, B7h.5, B7S1, B7x) is a transmembrane protein belonging to the B7/CD28 superfamily. The B7-H4 protein is expressed on activated T and B cells, monocytes and dendritic cells and may negatively regulate T cell immune responses.

B7-H4 mRNA is widely expressed, but B7-H4 protein is not expressed in most normal tissues. However, B7-H4 is overexpressed on the tumor cell surface of breast, ovarian and endometrial cancers. Breast cancer is the second largest malignancy in the world, and its prevalence is steadily increasing. About a quarter of female cancer patients are breast cancer patients. In 2019, there were 320,000 new cases of breast cancer in the United States, totaling about 3.8 million, and about 300,000 new cases of breast cancer in China each year, and the number of patients will reach 2.5 million in 2021. expected. Ovarian cancer and endometrial cancer are common malignant tumors of the female reproductive system. In China, about 50,000 women are diagnosed with ovarian cancer every year, and about 20,000 die from ovarian cancer, which ranks first among gynecologic malignancies in terms of mortality rate. With nearly 200,000 new cases each year, endometrial cancer ranks third among gynecologic malignancies leading to death.

B7-H4 is also overexpressed in non-small cell lung cancer and renal cancer. Lung cancer is one of the most common cancers, among which non-small cell lung cancer accounts for nearly 80% of lung cancers. Both in the world and in China, lung cancer has the highest morbidity and mortality rate. In 2018, there were about 2 million new lung cancer cases and 1.76 million deaths worldwide, with about 780,000 new cases and about 630,000 deaths each year in China. The prevalence of kidney cancer is low, with about 70,000 people suffering from kidney cancer in China every year.

B7-H4 has recently attracted attention as a new target for these tumors. B7-H4 antibody can act on tumor cells through multiple mechanisms, but the development direction of B7-H4 antibody is mainly focused on monoclonal antibody and ADC, and currently bispecific antibody therapy is do not have.

4-1BB (TNFRSF9, CD137) is a transmembrane protein belonging to the TNF receptor superfamily. 4-1BB is a multifunctional modulator for immune activity and a co-stimulatory molecule expressed on multiple types of cells. It is induced and expressed in activated T cells, NK cells, monocytes, dendritic cells, macrophages, tumor-associated vascular endothelial cells, and the like. Its ligand is 4-1BBL, which is mainly expressed on monocyte macrophages, DC cells, professional APC cells such as B cells, activated T cells, and some tumor cells. Anti-4-1BB agonistic antibodies can suppress tumors, but the development direction of 4-1BB antibodies is mainly focused on monoclonal antibodies and antibodies that simultaneously target PD-L1 and 4-1BB. . There are currently no bispecific antibodies targeting B7-H4 and 4-1BB in clinical development.

Bristol Myers Squibb (BMS)'s urelumab (BMS-663513) is a fully human IgG4 anti-4-1BB antibody and the first anti-4-1BB antibody to enter clinical trials. Although the first clinical results of urelumab were published in 2008 and an encouraging therapeutic effect was observed in some patients, the data showed that urelumab has hepatotoxicity and is related to target and dose. . Seriously, two patients died from hepatotoxicity in clinical trials, and clinical trials of urelumab as a single agent were discontinued. On the other hand, bispecific antibodies targeting PD-L1 and 4-1BB are currently only in the first stage of clinical trials, and overlap between the expression of PD-L1 and B7-H4 on tumor cells. Less is. Therefore, there is a need to develop safer and more effective bispecific antibodies that simultaneously target human B7-H4 and 4-1BB and can bind cynomolgus monkey B7-H4 and 4-1BB.

The problem to be solved by the present invention is to provide a safe and effective bispecific bispecific antibody that can simultaneously target human B7-H4 and 4-1BB and bind cynomolgus monkey B7-H4 and 4-1BB in the prior art. In order to overcome the shortage of biological antibodies, it is to provide a bispecific antibody, especially a bispecific antibody targeting B7-H4 and 4-1BB, and its application.

In order to solve the above problems, the present invention provides the following technical configurations.
A first aspect of the invention provides a bispecific antibody comprising an antigen binding domain targeting B7-H4 and an antigen binding domain targeting 4-1BB.
Preferably, said antigen binding domain targeting B7-H4 is HCDR1 as shown in SEQ ID NO: 16 as sequence, HCDR2 as shown in SEQ ID NO: 46 as sequence and HCDR3 as shown in SEQ ID NO: 84 as sequence; HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 59 as sequence and HCDR3 shown in SEQ ID NO: 98 as sequence; or HCDR1 shown in SEQ ID NO: 16 as sequence, HCDR2 shown in SEQ ID NO: 60 as sequence and preferably VH shown in SEQ ID NO: 142, VH shown in SEQ ID NO: 159 or VH shown in SEQ ID NO: 160 as sequence; more preferably heavy chain shown in SEQ ID NO: 174 as sequence , comprising the heavy chain set forth in SEQ ID NO: 191 or the heavy chain set forth in SEQ ID NO: 192;
More preferably, said antigen-binding domain targeting B7-H4 further comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of said LCDR1 is shown in SEQ ID NO: 112 or 113, and the amino acid sequence of said LCDR2 is shown in SEQ ID NO: 118; the LCDR3 amino acid sequence is shown in any of SEQ ID NOS: 131-133;
Even more preferably, said antigen binding domain targeting B7-H4 further comprises LCDR1 as shown in SEQ ID NO: 112 as sequence, LCDR2 as shown in SEQ ID NO: 118 as sequence and LCDR3 as shown in SEQ ID NO: 131 as sequence; LCDR1 shown in SEQ ID NO: 113, LCDR2 shown in SEQ ID NO: 118 as sequence and LCDR3 shown in SEQ ID NO: 132 as sequence; or LCDR1 shown in SEQ ID NO: 112 as sequence, LCDR2 shown in SEQ ID NO: 118 as sequence and LCDR3 as shown in SEQ ID NO: 133 as a sequence; preferably further comprises VL as shown in any of SEQ ID NOS: 166-169 as sequences; Including chains.
In bispecific antibodies such as those described above,
The antigen binding domain targeting said B7-H4 is preferably HCDR1 shown in SEQ ID NO: 16 as sequence, HCDR2 shown in SEQ ID NO: 46 as sequence and HCDR3 shown in SEQ ID NO: 84 as sequence, SEQ ID NO: 112 as sequence LCDR1 shown in SEQ ID NO: 118 as sequence and LCDR3 shown in SEQ ID NO: 131 as sequence; HCDR1 shown in SEQ ID NO: 23 as sequence, HCDR2 shown in SEQ ID NO: 59 as sequence and SEQ ID NO: 98 as sequence LCDR1 shown in SEQ ID NO: 113 as sequence, LCDR2 shown in SEQ ID NO: 118 as sequence and LCDR3 shown in SEQ ID NO: 132 as sequence; or HCDR1 shown in SEQ ID NO: 16 as sequence, sequence as HCDR2 shown in number 60 and HCDR3 shown in SEQ ID NO:84 as a sequence, LCDR1 shown in SEQ ID NO:112 as a sequence, LCDR2 shown in SEQ ID NO:118 as a sequence and LCDR3 shown in SEQ ID NO:133 as a sequence.
More preferably, said antigen binding domain targeting B7-H4 is VH shown in SEQ ID NO: 142 as sequence and VL shown in SEQ ID NO: 166 as sequence; VH shown in SEQ ID NO: 159 as sequence and sequence as sequence VH shown in SEQ ID NO: 160 as sequence and VL shown in SEQ ID NO: 168 as sequence; or VH shown in SEQ ID NO: 159 as sequence and VL shown in SEQ ID NO: 169 as sequence.
Even more preferably, said B7-H4 targeting antigen binding domain comprises a heavy chain as shown in SEQ ID NO: 174 as sequence and a light chain as shown in SEQ ID NO: 198 as sequence; light chain as shown in SEQ ID NO: 199 as sequence; heavy chain as shown in SEQ ID NO: 192 as sequence and light chain as shown in SEQ ID NO: 200 as sequence; or heavy chain as shown in SEQ ID NO: 191 as sequence and sequence as sequence It contains the light chain shown in number 201.
For the B7-H4-targeted antigen binding domain, the form is preferably in single VH, tandem VH, ScFv, Fab or IgG form. Therein said tandem VH is preferably a tandem of 2 or more VH's, eg a tandem of 2, 3 or 4 VH's. If in IgG form, the constant region it comprises is preferably derived from human IgG1 containing mutations L234A, L235A and P329G, or human IgG1 containing mutations L234A and L235A.
For antigen binding domains targeting 4-1BB as described above, comprising HCDR1, HCDR2 and HCDR3, said HCDR1, HCDR2 and HCDR3 are preferably as follows:
The HCDR1 sequence is shown in SEQ ID NO: 19 or its variant 1 or SEQ ID NO: 14, the HCDR2 sequence is shown in SEQ ID NO: 49 or its variant 2, SEQ ID NO: 51 or SEQ ID NO: 43, and the HCDR3 sequence is shown in SEQ ID NO:86 or variants thereof, SEQ ID NO:96, SEQ ID NO:89 or SEQ ID NO:81;
Mutations of said Variant 1 comprise one or more of T3I, S6N/R and Y7F; indicate either;
the mutations of Variant 2 include one or more of S1N/D, G2S/A, S3D/G, G5D/F/S/V and S6T/N/D; the sequence of Variant 2 is , preferably any of SEQ ID NOS: 47-48, SEQ ID NO: 50, SEQ ID NOS: 52-58 and SEQ ID NO: 61 in the sequence listing;
the mutations of Mutant 3 are G2R/D/A/K, S3A/T, S4G/N/A/T/H, E5T/V/M/G, T6A, D7G/S, H9Y/S, Y10H, Y11F, N12G/D and V13I/M/T; the amino acid sequence of said variant 3 is preferably SEQ ID NO: 85, SEQ ID NOS: 87-88 and SEQ ID NOS: 90-95 in the sequence listing. shown in one of the
In one embodiment, the 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 17, 47 and 85, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 48 and 86, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 49 and 87, respectively;
or comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 19, 50 and 88 as sequences, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 20, 51 and 89, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 52 and 90, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 49 and 90, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 21, 53 and 91, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 21, 54 and 92, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 55 and 93, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 49 and 86, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 49 and 94, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 22, 56 and 86, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 57 and 95, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 58 and 96, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 61 and 95, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 14, 43 and 81, respectively;
Preferably, said 4-1BB-targeted antigen binding domain comprises a heavy chain variable region, one or more of which sequences are shown in SEQ ID NOs: 143-157, 139, 284 or 161.
More preferably, said 4-1BB-targeting antigen binding domain comprises a heavy chain shown in SEQ ID NOS: 175-189, 193, 285 or 171 as a sequence.
Even more preferably, said 4-1BB-targeted antigen binding domain further comprises LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOS: 109, 118 and 128, respectively; more preferably the light chain shown in SEQ ID NO: 195 as a sequence.
In the present invention, the antigen-binding domain targeting 4-1BB as described above may be in any conventional form within the art, such as: single VH or tandem VH form, HCAb form or in the form of a ScFv; said tandem VH is preferably a tandem of 2 or more VHs, eg a tandem of 2, 3 or 4 VHs.
In a specific embodiment of the invention, said B7-H4-targeted antigen binding domain comprises LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOS: 112, 118 and 131, respectively, and SEQ ID NO: 16 as sequences, respectively. HCDR1, HCDR2 and HCDR3 shown in 46 and 84; and said 4-1BB-targeting antigen binding domain comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 109, 118 and 128 respectively as sequences, and SEQ ID NOs as sequences respectively. including HCDR1, HCDR2 and HCDR3 shown in 14, 43 and 81;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and wherein said 4-1BB-targeting antigen binding domain has LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOs: 109, 118 and 128, respectively, and HCDR1, HCDR2 as sequences set forth in SEQ ID NOs: 14, 43 and 81, respectively. and HCDR3;
said antigen-binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 17, 47 and 85, respectively;
said antigen-binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 48 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and the 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 49 and 87, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 19, 50 and 88, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 20, 51 and 89, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and the 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 52 and 90, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 21, 53 and 91, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 21, 54 and 92, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 19, 55 and 93, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 49 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 49 and 94, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 22, 56 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 57 and 95, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 17, 47 and 85, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 48 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 49 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 49 and 94, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 61 and 95, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 48 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and said 4-1BB-targeted antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 19, 58 and 96, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 57 and 95, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domains are shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 49 and 90, respectively.

For bispecific antibodies according to the invention, in specific embodiments of the invention:
said antigen domain targeting B7-H4 is in the form of IgG and said antigen binding domain targeting 4-1BB is in the form of a single VH;
Alternatively, the antigen domain targeting B7-H4 is in the form of IgG and the antigen binding domain targeting 4-1BB is in the form of ScFv;
Alternatively, said B7-H4-targeting antigen domain is in the form of IgG and said 4-1BB-targeting antigen binding domain is in the form of a tandem of two or three VHs;
Alternatively, the B7-H4-targeting antigen domain is in the form of Fab, and the 4-1BB-targeting antigen-binding domain is in the form of HCAb or in the form of HCAb and VH (HCAb-VH form). ;
Alternatively, the antigen domain targeting B7-H4 is in the form of Fab, the antigen binding domain targeting 4-1BB is in the form of VH, and the VH form is preferably a single VH, two Or in the form of a tandem of 3 VHs.

More specifically,
Said bispecific antibody is of the form:
(1) ScFv is connected to the C-terminus of IgG, and VH of said ScFv is connected to said C _- terminus; and polypeptide chain 2 is shown in the formula: VH _B7-H4 -CH1-hinge-CH2-CH3-linker-VH _4-1BB -linker-VL _4-1BB ;
(2) VH or tandem VH is connected to the C-terminus of IgG; preferably, the _polypeptide chain 1 of the bispecific antibody of this form is represented by the formula: : VH _B7-H4 -CH1-hinge-CH2-CH3-linker-(VH _4-1BB )n-;
(3) Fab is attached to the N-terminus of the HCAb; preferably, polypeptide chain 1 of the bispecific antibody of this form is represented by the formula: VH _B7-H4 -CH1, and polypeptide chain 2 is represented by the formula: VL _{B7- H4} -CL-linker-VH _4-1BB -linker-CH2-CH3 shown;
(4) Fab is connected to the N-terminus of HCAb _and VH is connected to the C-terminus of said Fab; , polypeptide chain 2 is shown in the formula: VL _B7-H4 -CL-linker-VH _4-1BB -linker-CH2-CH3-VH _4-1BB ;
or (5) the N-termini of the two CH2s of Fc are each connected to a Fab and a VH or VH in tandem; preferably, the form of the bispecific antibody comprises three polypeptide chains:
Polypeptide chain 1 is shown in the formula: VL _B7-H4 -CL;
Polypeptide chain 2 is represented by the formula: VH _B7-H4 -CH1-hinge-CH2-CH3;
Polypeptide chain 3 has the formula: VH _4-1BB -linker-CH2-CH3 or (VH _4-1BB )n-linker-CH2-CH3;
Among them, the sequences of said linkers connecting different domains are the same or different.
wherein: the Fc of said IgG or HCAb contains a mutation, preferably one of the following mutations:
1) For IgG1, L234A and L235A by EU number, optionally further comprising P329G, YTE or DHS; mutated, Q at position 311 mutated to H and N at position 434 mutated to S;
2) for IgG1, deletion of positions 233-235 by EU number;
3) for IgG1, L234A and G237A by EU number;
4) For IgG4, S228P and FALA by EU number;
5) For IgG1, N297A by EU number.
The hinge as described above and the linker as described above may be conventional in the art, preferably the linker as described above is selected from the group consisting of SEQ ID NOS: 241-261, 282 and 288-289; in:
A linker of form (1) preferably comprises the sequence shown in SEQ ID NO:245;
Linkers of form (2) preferably comprise sequences shown in any of SEQ ID NOs:243, 245-247 and SEQ ID NOs:288-289;
A linker of form (3) preferably comprises the sequence shown in SEQ ID NO:250;
A linker of form (4) preferably comprises the sequence shown in SEQ ID NO:282;
A linker of form (5) preferably comprises the sequence shown in SEQ ID NO:245.
In one preferred embodiment of the present invention, the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 198 and the amino acid sequence of said polypeptide chain 2 is shown in any of SEQ ID NOs: 202-215;
or the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 201 and the amino acid sequence of said polypeptide chain 2 is shown in SEQ ID NOs: 217, 230, 238, 240, 226, 262 or 239;
Alternatively, the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 200, and the amino acid sequence of said polypeptide chain 2 is any of SEQ ID NOS: 218-225, 235-237, 263-268, 274-281, 286 and 287 to show;
Alternatively, the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 201, the amino acid sequence of said polypeptide chain 2 is shown in SEQ ID NO: 227, and the amino acid sequence of said polypeptide chain 3 is shown in SEQ ID NO: 228, 229, 231 or shown at 232;
Alternatively, the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 233 and the amino acid sequence of said polypeptide chain 2 is shown in any of SEQ ID NOs: 234, 269-273.
A second aspect of the invention provides an isolated nucleic acid encoding the bispecific antibody of the first aspect.
A third aspect of the invention provides an expression vector comprising the isolated nucleic acid of the second aspect.
A fourth aspect of the invention comprises an expression vector according to the third aspect; and provides a host cell, preferably a prokaryotic or eukaryotic cell.
A fifth aspect of the present invention provides a method of making a bispecific antibody comprising culturing the host cell of the fourth aspect and obtaining said bispecific antibody from the culture. .
A sixth aspect of the invention provides a pharmaceutical composition comprising a bispecific antibody according to the first aspect of the invention.
A seventh aspect of the present invention is the use of the bispecific antibody according to the first aspect, the pharmaceutical composition according to the sixth aspect, to prevent and/or prevent 4-1BB and/or B7-H4 related diseases or provide use in making drugs for treatment.
Among them, said disease is preferably cancer, preferably breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumor, Bile duct cancer, gallbladder cancer, bladder cancer, sarcoma or colorectal cancer. Preferably, said cancer is breast cancer, ovarian cancer, endometrial cancer, renal cancer or cholangiocarcinoma. More preferably, said cancer is breast cancer.
An eighth aspect of the invention provides a chimeric antigen receptor comprising the bispecific antibody of the first aspect.
A ninth aspect of the present invention provides an antibody drug conjugate comprising a cytotoxic agent and a bispecific antibody according to the first aspect; preferably said cytotoxic agent is MMAF or MMAE.
A tenth aspect of the present invention is the bispecific antibody according to the first aspect, the chimeric antigen receptor according to the eighth aspect, the antibody-drug conjugate according to the ninth aspect and/or the sixth aspect. comprising a pharmaceutical composition according to the form of
Preferably, a reagent kit is provided further comprising (i) a device for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
An eleventh form of the present invention is a drug kit comprising kit A and kit B,
The kit A comprises the bispecific antibody according to the first aspect, the chimeric antigen receptor according to the eighth aspect, the antibody-drug conjugate according to the ninth aspect and/or the sixth aspect. comprising a pharmaceutical composition;
Said kit B comprises another anti-tumor antibody or a pharmaceutical composition comprising said other anti-tumor antibody, and/or a hormone preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic agent A drug kit is provided that includes one or more of the group consisting of toxic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.
A twelfth aspect of the present invention is a method of diagnosing, treating and/or preventing a 4-1BB and/or B7-H4 mediated disease or disorder, comprising:
A therapeutically effective amount of the bispecific antibody according to the first aspect, the chimeric antigen receptor according to the eighth aspect, the antibody-drug conjugate according to the ninth aspect, or the sixth aspect in a patient in need thereof. A method comprising administering a pharmaceutical composition as described or treating a patient in need thereof with a drug kit according to the eleventh aspect.
Among them, said disease or disorder is preferably a tumor, preferably breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumor, bile duct. cancer, gallbladder cancer, bladder cancer, sarcoma or colorectal cancer; preferably said cancer is breast cancer, ovarian cancer, endometrial cancer, renal cancer or cholangiocarcinoma; more preferably said cancer is breast cancer.
An eleventh aspect of the present invention is a method for immunodetecting or measuring 4-1BB or B7H4, comprising the bispecific antibody according to the first aspect and the chimeric antigen receptor according to the eighth aspect. preferably, said detection is not for diagnostic and/or therapeutic purposes.
A fourteenth aspect of the present invention provides a patient in need thereof with the bispecific antibody according to the first aspect, the chimeric antigen receptor according to the eighth aspect, and the antibody-drug conjugate according to the ninth aspect. or administering, respectively, a pharmaceutical composition according to the sixth aspect and a second therapeutic agent;
Said second therapeutic agent is preferably another anti-tumor antibody or a pharmaceutical composition comprising said other anti-tumor antibody, and/or a hormone preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent , oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

The numbers in "mutant 1", "mutant 2" and "mutant 3" in the present invention have no special meaning and are only used to distinguish between the same terms.

The B7-H4x4-1BB bispecific antibodies of the invention are specific B7-H4x4-1BB bispecific antibodies that have one or two or three sites that bind to 4-1BB. the two protein functional domains of the bispecific antibody both have good binding activity to cynomolgus monkeys.

The positive advance effects of the present invention are as follows:
1. The 4-1BB antibody of the present invention is a completely new, fully human antibody containing only a "heavy chain" and has the activity of binding to human 4-1BB and cynomolgus monkey 4-1BB. The feature of this 4-1BB heavy chain antibody, which is only half the size of a conventional IgG antibody and does not contain a light chain, allows this antibody to be used as a bispecific antibody, where light chain mismatches and Solved the quantification problem.
2. The B7-H4 antibody of the present invention is a completely new fully human antibody and has the activity of binding to human B7-H4 and cynomolgus monkey B7-H4.
3. The bispecific antibodies of the invention have one, two or three sites that bind 4-1BB, optimizing the activity of the 4-1BB terminus.
4. The present invention specifically binds tumor cells by targeting B7-H4 and reduces toxicity due to 4-1BB activation.
5. The present invention is a bispecific antibody structure with a human Fc fragment, which retains the binding action of Fc and FcRn, thereby having a long half-life.

In FIG. 1, (A) shows FACS detection of in vitro binding of B7-H4 monoclonal antibody in SK-BR-3 cell line highly expressing B7-H4; (B) shows B7-H4 monoclonal antibody. (C) shows FACS detection of in vitro binding in CHO-K1-cynomolgus monkey B7-H4 cell lines overexpressing cynomolgus monkey B7-H4 of B7-H4 monoclonal antibody; FACS detection of in vitro binding in the K1-mouse B7-H4 cell line. Figures 2-(A)-(I) show FACS detection of in vitro binding of the 4-1BB monoclonal antibody in a CHO-K1-human 4-1BB cell line that highly expresses human 4-1BB. Ditto. Ditto. Figures 3-(A)-(H) show FACS detection of in vitro binding in the CHO-K1-cynomolgus 4-1BB cell line, which highly expresses the 4-1BB monoclonal antibody cynomolgus 4-1BB. Ditto. Ditto. Figures 4-(A)-(E) show the function of the 4-1BB monoclonal antibody to activate the 4-1BB pathway and induce and activate T cells. Ditto. 5-(A)-(I) are structural schematics of the B7-H4×4-1BB bispecific molecule. Ditto. Ditto. Ditto. Ditto. FIGS. 6-(A)-(L) show FACS detection of in vitro binding of B7-H4×4-1BB bispecific antibody in CHO-K1 cell line overexpressing human 4-1BB. Ditto. Ditto. Ditto. FIGS. 7-(A)-(C) show FACS detection of in vitro binding of B7-H4×4-1BB bispecific antibody in CHO-K1 cell line overexpressing cynomolgus monkey 4-1BB. FIGS. 8-(A)-(K) show FACS detection of in vitro binding of the B7-H4×4-1BB bispecific antibody in the SK-BR-3 cell line highly expressing B7-H4. Ditto. Ditto. Ditto. Figure 9-(A)-(E) shows T cell activation experiments in the presence of a cell line SK-BR-3 that highly expresses the B7-H4×4-1BB bispecific antibody B7-H4. and the release of the cytokine IFN-γ. Ditto. FIG. 10-(A)-(N) are T cell activation experiments in the presence of the cell line SK-BR-3, which highly expresses the B7-H4×4-1BB bispecific antibody B7-H4. and release of the cytokine IL-2. Ditto. Ditto. Ditto. Ditto. Ditto. FIGS. 11-(A)-(B) show that the activation of B7-H4×4-1BB bispecific antibody against T cells is independent of the presence of FcγR. FIGS. 12-(A)-(B) show FACS detection of B7-H4 expression levels on tumor cell surfaces. FIGS. 13-(A)-(H) show that the activation of B7-H4×4-1BB double antibody against T cells is specifically dependent on B7-H4 expression. Ditto. Ditto. Figures 14-(A)-(H) show the serum stability of the B7-H4x4-1BB double antibodies PR003334, PR003335 and PR004282. Ditto. Ditto. Figures 15-(A)-(C) show the in vitro binding of the B7-H4x4-1BB double antibody PR004282 to human, monkey and mouse 4-1BB. Figures 16-(A)-(C) show the in vitro binding of the B7-H4x4-1BB double antibody PR004282 to human, monkey and mouse B7H4. FIG. 17 shows in vitro simultaneous binding of the B7-H4×4-1BB double antibody PR004282 to SK-BR-3 and CHO-K1/4-1BB. Figures 18-(A)-(B) show the in vitro binding of the B7-H4x4-1BB double antibody PR004282 to in vitro activated human, monkey Pan T cells. Figures 19-(A)-(B) show the results of cross-reactivity between the B7-H4x4-1BB double antibody PR004282 and B7 family members or other members of the TNFR family. Figures 20-(A)-(C) show the results of the ADCC effect of the B7-H4x4-1BB double antibody PR004282. Figures 21-(A)-(C) show the results of detection of non-specific cytokine release by the B7-H4x4-1BB bispecific antibody PR004282. Figures 22-(A)-(F) show pharmacokinetic data in wild-type mice for B7-H4x4-1BB bispecific antibodies PR003334, PR003335 and PR004282. Ditto. Figure 23 shows pharmacokinetic data (single dose) in normal monkeys for the B7-H4x4-1BB bispecific antibody PR004282. Figures 24-(A)-(B) show the anti-tumor efficacy of the B7-H4x4-1BB bispecific antibody PR003334 in the BALB/c-hCD137/CT26-B7-H4 mouse model. Figures 25-(A)-(B) show the anti-tumor efficacy of the B7-H4x4-1BB bispecific antibody PR003338 in the MDA-MB-468 xenograft mouse model. Figures 26-(A)-(B) show the anti-tumor efficacy of the B7-H4x4-1BB bispecific antibody PR004282 in the OVCAR3 xenograft mouse model. FIG. 27-(A)-(I) Anti-tumor efficacy and memory immunity of B7-H4×4-1BB bispecific antibody PR004282 in BALB/c-hCD137/CT26-B7-H4 mouse model. Effect results are shown. Ditto. Ditto. Ditto. Figures 28-(A)-(B) show the specific results of the anti-tumor efficacy of the B7-H4x4-1BB bispecific antibody PR004282.

The present invention will be further described in the form of examples below, but the scope of the present invention is not thereby limited to the above examples. Experimental methods for which specific conditions are not described in the following examples are selected according to usual methods and conditions or according to product instructions.
As used herein, the term "antibody" generally refers to a protein comprising an antigen-binding portion, and the antigen-binding portion optionally employs a conformational scaffold or framework portion capable of promoting antibody-antigen binding. be able to. Typically, it may comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. For example, a "heavy chain antibody" in this application does not include a VL region, but only a VH region. The VH or VL region can be further divided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conservative, termed framework regions (FR). Each VH or VL can be composed of three CDRs and four FR regions, arranged from amino-terminus to carboxyl-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigen. Examples of antibodies include full length antibodies, heavy chain antibodies (HCAb), antigen binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and/or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins. Not limited.

In this application, the term "variable" generally refers to the fact that some partial variations in the sequence of variable domains of antibodies are so strong that they form specific bindings of various specific antibodies to specific antigens. Point. However, the variability is not evenly distributed throughout the variable regions of antibodies. Concentrated on three segments of the light and heavy chain variable regions, called CDRs or hypervariable regions (HVRs), the FRs are the more highly conservative portions of the variable region. The variable domains of naturally occurring heavy and light chains each contain four FR regions, mostly adopting a β-sheet structure, connected by three CDRs to form loop connections, and in some cases a β-sheet structure. forms part of The CDRs in each chain are contiguous to each other via the FR region and together with the CDRs from other chains form the antigen-binding site of the antibody, and the constant region is not directly involved in antibody-antigen binding, but the antibody depends on the antibody. It exhibits different effect functions such as cytotoxicity.

As used herein, the term "fully human antibody" generally refers to an antibody expressed by transferring all of the genes encoding human antibodies to a genetically engineered antibody gene-deficient animal. All portions of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Wholly human-derived antibodies can greatly reduce immune side reactions in the human body caused by heterologous antibodies. Methods of obtaining fully human-derived antibodies in the art include phage display technology and transgenic mouse technology.

As used herein, the term "nucleic acid" refers to DNA and RNA molecules. It may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

As used herein, the term "specifically binds" generally means that an antibody binds to an epitope via its antigen-binding domain, and the binding involves some degree of interaction between the antigen-binding domain and the epitope. Complementarity is required. According to this definition, an antibody is said to "specifically bind" an antigen if it is more likely to bind an epitope through its antigen-binding domain than to a random unrelated epitope. .

In this application, the term "Fab" generally refers to an antigen in a conventional antibody (e.g. IgG) comprising the antibody heavy chain variable region VH, light chain variable region VL, heavy chain constant region domain CH1 and light chain constant region CL. refers to the part that connects with In a normal antibody, the C-terminus of VH is linked to the N-terminus of CH1 to form the heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form the light chain, and the C-terminus of CH1 is It then joins the heavy chain hinge region and other constant region domains to form a heavy chain. In some embodiments, "Fab" also refers to mutant structures of Fab. For example, in one embodiment, the C-terminus of VH and the N-terminus of CL are joined to form one polypeptide chain, and the C-terminus of VL and the N-terminus of CH1 are joined to form another polypeptide chain. , forming a structure of Fab (cross VH/VL); in certain embodiments, the CH1 of the Fab is not attached to the hinge region, the C-terminus of CL is attached to the hinge region of the heavy chain, and the Fab (cross Fd/LC ).

In the present application, the term "VH" usually refers to the heavy chain variable region VH domain of an antibody, i.e. it may be the heavy chain variable region VH of a human or other animal conventional antibody (H2L2 structure), It may be the heavy chain variable region VHH of an animal heavy chain antibody (HCAb structure) such as Camelidae, or the heavy chain variable region of a fully human-derived heavy chain antibody (HCAb structure) produced using Harbor HCAb transgenic mice. It may be the region VH.

Kabat definitional rules based on sequence variability (see Kabat et al., Immunological Protein Sequences, 5th Edition, National Institutes of Health, Bethesda, Md. (1991)) and the definition of structural loop regions. The CDRs of an antibody can be defined in a variety of ways, such as the position-based Chothia definition rules (see A1-Lazikani et al., Jmol Biol 273:927-481997). The present application can also determine amino acid residues in variable domain sequences and full-length antibody sequences using the Combined definition rules, including the Kabat and Chothia definitions (Table 1). In the present invention, sequences are determined based on the Chothia definition.

Here, Laa-Lbb can refer to the amino acid sequence from position aa (Chothia coding rules) to position bb (Chothia coding rules) from the N-terminus of the antibody light chain, and Haa-Hbb is the antibody weight. The amino acid sequence from position aa (Chothia coding rules) to position bb (Chothia coding rules) from the N-terminus of the chain can be referred to. For example, L24-L34 can refer to the amino acid sequence from position 24 to position 34 based on the Chothia coding rules from the N-terminus of the antibody light chain, and H26-H32 refers to the antibody heavy chain N-terminus to the Chothia coding rules. can refer to the amino acid sequence from position 26 to position 32 based on The present invention uses the Chothia coding rules to define antibody CDRs.

Antibody Fc domain-mediated effector functions, such as ADCC and CDC, also have very important biological functions, and different IgG subtypes have different ADCC or CDC functions, such as strong ADCC for IgG1 and IgG3. and have a CDC effect, and IgG2 and IgG4 are relatively weak. Alteration of the binding ability of Fc and Fc receptors by amino acid mutation or modification can also modulate the intrinsic effector function of Fc. For example, the "LALA" double mutant (L234A/L235A) in IgG1 can significantly reduce affinity for FcγRIIIA (CD16A) and reduce ADCC effects. The P329G mutation can also significantly reduce binding to multiple Fcγ receptors (Schlothauer T, Herter S, Koller CF, Grau-Richards S, Steinhart V, Spick C, Kubbies M, Klein C , Umana P, Mossner E. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions.Protein EngDes Sel.2016 Oct, 2 9(10):457-466.doi:1093/protein/gzw040.Eub 2016. Aug 29. PubMed PMID: 27578889). In this application, in order to reduce the binding of the B7-H4x4-1BB bispecific antibody to the Fcγ receptor, the Fc of these antibodies has the "LALA" double mutant (L234A/L235A) or "LALAPG" A triple mutant (L234A/L235A/P329G) was introduced.

Example 1. Antibody Sequence Analysis, Expression Purification, and Physicochemical Characterization Analysis 1.1 Antibody Sequence Analysis and Optimization derived from events such as fragment genetic rearrangements and somatic hypermutation; Derived from events such as mutations. Gene rearrangements and somatic hypermutation are major factors in increasing antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but the overall similarity is high. by computational methods such as IMGT/DomainGapAlign (http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) or NCBI/IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/) , it is possible to deduce possible germline gene fragments if gene rearrangements occur from the antibody variable domain sequences.

A protein or polypeptide amino acid chain may introduce chemical modifications, called post-translational modifications (PTMs), after being translated and synthesized in the cell. Some PTM sites are very conservative for antibodies, for example the conservative amino acid Asparagine Asn at position 297 (EU number) of the constant domain of the human IgG1 antibody, which is commonly glycosylated to form sugars. Forming chains, this carbohydrate structure is crucial to antibody structure and related effect functions. However, when PTMs are present in the variable domains of antibodies, particularly in antigen-binding domains such as CDRs, the presence of these PTMs can have a significant impact on antigen binding, and also change the physicochemical properties of antibodies. may bring about. For example, glycosylation, deamidation, isomerization, oxidation, etc. can all increase the instability or heterogeneity of the antibody molecule, thereby increasing the difficulty and risk of antibody development. Therefore, avoidance of some potential PTMs is of great importance for the development of therapeutic antibodies. With accumulating experience, it was discovered that some PTMs are highly associated with amino acid sequence composition, particularly the 'pattern' of adjacent amino acid composition, which allows the identification of potential PTMs from the primary amino acid sequence of a protein. can be predicted. For example, a sequence pattern of NxS/T (asparagine at position 1, any amino acid except proline at position 2, serine or threonine at position 3) predicts an N-linked glycosylation site. Amino acid sequence patterns that cause PTMs can be derived from germline gene sequences, for example the human germline gene fragment IGHV3-33 has a naturally occurring glycosylation pattern NST in the FR3 region; It may also result from mutation. For example, NGS or NLT is a glycosylation site, NS is a deamidation site, and DG can cause asparagine isomerization.

Amino acid mutations can disrupt the amino acid sequence pattern of PTMs and further reduce or eliminate the formation of certain PTMs. There are different mutation design methods depending on the antibody sequence and PTM sequence pattern. One method is to replace "hot spot" amino acids such as N or S in NS mode with amino acids with similar physicochemical properties (such as mutating N to Q). If the PTM sequence pattern is derived from somatic hypermutation and is not present in the germline gene sequence, another approach is to replace the sequence pattern with the corresponding germline gene sequence. In practical operation, multiple mutation design methods can be employed for the same PTM sequence pattern.

1.2 Antibody Expression and Purification This example uses techniques such as mammalian host cells (e.g., human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derivatives), instantaneous transfection expression and affinity capture isolation. A general method for producing antibodies was introduced. The method applies to targeting antibodies that contain an Fc region. A targeting antibody can be composed of one or more protein polypeptide chains and can be derived from one or more expression plasmids.

The amino acid sequences of the antibody polypeptide chains are converted to nucleotide sequences by codon optimization methods; the encoded nucleotide sequences are synthesized and cloned into an expression vector compatible with the host cell. Plasmids encoding antibody polypeptide chains are co-transfected into mammalian host cells in specific proportions and conventional recombinant protein expression and purification techniques are used to produce recombinant antibodies with correct folding and polypeptide chain assembly. can be obtained. Specifically, FreeStyle ^™ 293-F cells (Thermo, #R79007) were expanded in FreeStyle ^™ F17 Expression Medium medium (Thermo, #A1383504). Cell concentration was adjusted to 6-8×10 ⁵ cells/ml and cultured in a 37° C., 8% CO ₂ shaker for 24 hours prior to initiation of flash transfection, with a cell concentration of 1.2×10 ⁶ cells/ml. ml. Prepare 30 ml of cultured cells. Plasmids encoding antibody polypeptide chains were mixed at a constant ratio, and a total of 30 μg of plasmid (ratio of plasmid to cells: 1 μg:1 ml) was dissolved in 1.5 ml of Opti-MEM reduced serum medium (Thermo, #31985088). and filtered and sterilized with a 0.22 μm filtration membrane. Further, 1.5 ml of Opti-MEM was taken and dissolved in 120 μl of 1 mg/ml PEI (Polysciences, #23966-2) and allowed to stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop the plasmid-PEI mixed solution while shaking the culture bottle, and incubate at 37°C, 8% CO ₂ shaker for 5 days. Cell viability was observed after 5 days. The culture was harvested and centrifuged at 3300 g for 10 minutes, after which the supernatant was taken; the supernatant was then centrifuged at high speed to remove impurities. Equilibrate a gravity column (Bio-Rad, #7311550) containing MabSelect ^™ (GE Healthcare, #71-5020-91) with PBS pH 7.4 buffer and wash 2-5 column volumes. Pass the supernatant sample through the column; wash the column with 5-10 column volumes of PBS buffer, elute the protein of interest with 0.1 M glycine, pH 3.5, followed by Tris-HCl, pH 8.0. and finally concentration-exchanged into PBS buffer or a buffer containing other components in an ultrafiltration tube (Millipore, #UFC 901024) to obtain a purified recombinant antibody solution. Finally the concentration is measured with a NanoDrop (Thermo, NanoDrop ^™ One), aliquoted and saved in reserve.

1.3 Protein Purity and Multimer Analysis by SEC-HPLC In this example, analytical molecular size exclusion chromatography (SEC) was used to analyze protein samples for purity and polymeric form. An analytical chromatography column TSKgel G3000 SWxl (Tosoh Bioscience, #08541, 5 μm, 7.8 mm × 30 cm) was connected to a high-pressure liquid chromatography HPLC (Agilent Technologies, Agilent 1260 Infinity II), and PBS buffer was used. Equilibrate at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μg) is filtered through a 0.22 μm filter membrane and injected into the system, and the HPLC program is set up: run the sample in PBS buffer through the chromatography column at a flow rate of 1.0 ml/min and Time is 25 minutes. HPLC produces an analytical report, reporting the retention times of different molecular size components within the sample.

1.4 Determination of Thermal Stability of Protein Molecules by DSF Differential Scanning Fluorimetry (DSF) is a common high-throughput method for measuring the thermal stability of proteins. A real-time fluorescence quantitative PCR instrument is used to monitor changes in fluorescence intensity of dyes bound to unfolded protein molecules, reflecting the process of protein denaturation and reflecting the thermal stability of protein molecules. In this example, the DSF method was used to measure the thermal denaturation temperature (Tm) of protein molecules. Add 10 μg protein to a 96-well PCR plate (Thermo, #AB-0700/W) followed by 2 μl 100× diluted dye SYPRO™ (Invitrogen, #2008138) for a final volume of 40 μl per well. Buffer was added as follows. The PCR plate was sealed, placed in a real-time fluorometric PCR machine (Bio-Rad CFX 96 PCR System) and incubated at 25°C for 5 min, followed by a 0.2°C/0.2 min gradient from 25°C. The temperature was gradually raised to 95°C and lowered to 25°C at the end of the test. Data analysis was performed using Bio-Rad CFX Maestro software using the FRET scan mode to calculate the Tm of the samples.

Example 2. Acquisition of Anti-B7-H4 Fully Human Antibodies Harbor H2L2 mice (Harbor Antibodies BV) are transgenic mice carrying a human immunoglobulin immune library and the antibodies produced contain fully human antibody variable domains and rat constant domains. have.

Multiple rounds of immunization of Harbor H2L2 mice with soluble recombinant human B7-H4-ECD-mFc fusion protein (Sino Biological, #10738-H05H) or with CHO-K1 cells overexpressing human B7-H4. Harbor H2L2 mice were immunized for multiple rounds after transfection with mCD40L using immunogens and mixed with an immunogenic adjuvant to form an immunogenic reagent.
After detecting that the B7-H4-specific antibody titer in mouse serum reached a certain level, mouse spleen cells were taken out. Screening is performed by hybridoma fusion technology, B cell cloning technology and phage library technology, and the monoclonal antibodies obtained by screening are further identified and tested for parameters such as binding ability to human B7-H4 and cynomolgus monkey B7-H4. Based on this, two clones 80C8-2E9 and 1025_B-1H11 were selected. Nucleotide sequences encoding the variable domains of antibody molecules and corresponding amino acid sequences are obtained by conventional sequencing means. Sequence analysis and optimization of the candidate antibody molecules were then performed and several variant sequences were obtained. The VL and VH sequences of the antibody were fused with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain a recombinant whole human antibody molecule.
The anti-B7-H4 recombinant whole human IgG antibodies in this example are shown in Table 2.

Also, according to a patent inquiry (from patent WO2016040724A1), 1D11. Control Antibody 1, also called v1.9varC2, was designed to serve as a control in subsequent experiments:

2.1 FACS Detection of Cellular Binding Ability of Anti-Human B7-H4 H2L2 Monoclonal Antibody This example demonstrates the in vitro binding of anti-human B7-H4 H2L2 monoclonal antibody to human/cynomolgus/mouse B7-H4. for studying activity. CHO-K1 cell line overexpressing cynomolgus monkey B7-H4 (CHO-K1/cyno B7-H4, manufactured by HARBOUR BIOMED), CHO-K1 cell line overexpressing mouse B7-H4 (CHO-K1/m B7-H4 , HARBOUR BIOMED), and a cell line SK-BR-3 (ATCC ^{(registered trademark)} HTB-30) that highly expresses human B7-H4, were used to perform antibody binding experiments at the cellular level. Briefly, CHO-K1/cyno B7-H4, CHO-K1/m B7-H4 or SK-BR-3 cells were digested and resuspended in PBS containing 2% BSA. Cell densities were adjusted to 1×10 ⁶ cells/mL respectively. After seeding 96-well V-bottom plates (Corning, #3894) with 100 μL cells/well, 100 μL/well of antibody to be measured diluted in a 3-fold gradient of 2-fold the final concentration was added. Cells were placed at 4°C and incubated away from light for 2 hours. Cells were then washed twice by adding 100 μL/well of pre-chilled PBS containing 2% BSA, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded. Additionally 100 μL/well fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-06, diluted 1:500) at 4° C. away from light. Incubated for 1 hour. Cells were washed twice by adding 100 μL/well pre-chilled PBS containing 2% BSA, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded. Finally, cells were resuspended with 200 μL/well pre-chilled PBS containing 2% BSA and fluorescence signal values were read using an ACEA Novocyte 3000 flow cytometer.

As shown in FIG. 1-(A), in vitro binding of B7-H4 antibody in SK-BR-3 cell line highly expressing B7-H4; , superior to control antibody 1.
In vitro binding of B7-H4 antibody in CHO-K1-cynomolgus monkey B7-H4 cell line highly expressing cynomolgus monkey B7-H4; Cell line binding activity is superior to control Antibody 1.
As shown in FIG. 1-(C), in vitro binding of B7-H4 antibody in CHO-K1-mouse B7-H4 cell line highly expressing mouse B7-H4; PR002410 and CHO-K1-mouse B7-H4 cell line was not significantly different from control antibody 1.

Example 3.4 Acquisition of 1BB Pan-Human Antibody 3.1 Acquisition of Anti-4-1BB Pan-Human HCAb Antibody Harbor HCAb mice (Harbor Antibodies BV, WO2010/109165A2) are transgenic with a human immunoglobulin immune library. It is murine and can produce heavy chain-only antibodies that are only half the size of conventional IgG antibodies. The antibody produced has only human antibody heavy chain variable domains and mouse Fc constant domains.

Harbor HCAb mice were immunized multiple rounds with soluble recombinant human 4-1BB-Fc fusion protein (ChemPartner) or NIH-3T3 cells overexpressing human 4-1BB (ChemPartner). After detecting that the 4-1BB-specific antibody titer in the mouse serum reached a certain level, the mouse spleen cells were taken out to separate B cells, and CD138-positive plasma cells were sorted using BD FACS AriaII Cell Sorter. and a human 4-1BB antigen-positive B cell population. Human VH genes were amplified from plasma cells using conventional molecular biology tools, and the amplified human VH gene fragments were constructed into the mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc region sequences. . A plasmid-transfected mammalian host cell (eg, human embryonic kidney HEK 293) was expressed to obtain a total human-derived HCAb antibody supernatant. Binding of the HCAb antibody supernatant to CHO-K1 cells CHO-K1/hu4-1BB highly expressing human 4-1BB was tested using FACS to identify positive HCAb antibodies. These HCAb antibodies were further identified and preferably several candidate HCAb antibody molecules were selected based on parameters such as ability to bind human 4-1BB, ability to bind cynomolgus monkey 4-1BB, ability to activate T cells. Subsequent sequence analysis and optimization of candidate HCAb antibody molecules resulted in several variant sequences. The HCAb antibody VH sequence and human IgG1 heavy chain Fc sequence were fused and expressed to obtain a fully human-derived recombinant HCAb antibody molecule. Here, PR004469 is a PTM mutant of PR001838, and PR007381 is a germlined mutant of PR004469, and specific mutation sites are shown in Table 5.

3.2 Acquisition of Anti-4-1BB Fully Human H2L2 Antibodies Harbor H2L2 mice (Harbor Antibodies BV) are transgenic mice carrying a human immunoglobulin immune library and antibodies produced with fully human antibody variable domains. It has a rat constant domain. Harbor H2L2 mice were subjected to multiple rounds of immunization with soluble recombinant human 4-1BB-Fc fusion protein. After detecting that the 4-1BB-specific antibody titer in the mouse serum reached a certain level, the spleen cells of the mouse were taken out and fused with myeloma cell lines to obtain hybridoma cells. After screening and cloning, a hybridoma cell line expressing an anti-4-1BB monoclonal antibody molecule was isolated. Nucleotide sequences encoding antibody molecule variable domains and corresponding amino acid sequences were obtained using conventional hybridoma sequencing tools. Sequence analysis and optimization of the candidate antibody molecules were then performed and several variant sequences were obtained. The VL and VH sequences of the antibody were fused to the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain a recombinant whole human antibody molecule.

Also, according to the patent inquiry, a control antibody was designed to serve as a control in subsequent experiments:

3.3 FACS Detection of Cellular Binding Ability of Anti-Human 4-1BB Monoclonal Antibody It is intended to study CHO-K1 cell line overexpressing human 4-1BB (CHO-K1-hu 4-1BB, Genescript), CHO-K1 cell line overexpressing cynomolgus monkey 4-1BB (CHO-K1-cyno 4-1BB, Genescript ) was used to perform antibody binding experiments at the cellular level. Briefly, the cells CHO-K1-hu 4-1BB and CHO-K1-cyno 4-1BB cells were digested, resuspended in DMEM complete medium and the cell density was adjusted to 1×10 ⁶ cells/mL respectively. . After seeding 96-well V-bottom plates (Corning, #: 3894) with 100 μL cells/well, 100 μL/well of antibody to be measured diluted in a 3-fold gradient of 2-fold the final concentration was added. Cells were placed at 4°C and incubated for 1 hour away from light. Cells were then washed twice by adding 100 μL/well pre-chilled PBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. In addition, 100 μL/well of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #: 109-545-06, diluted 1:500), at 4° C., protected from light. and incubated for 30 minutes. Cells were washed twice with 100 μL/well pre-chilled PBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended with 200 μL/well of pre-chilled PBS and fluorescence emission signal values were read using BD FACS CANTOII.

FIG. 2-(A)-(I): In vitro binding of 4-1BB antibody in CHO-K1-human 4-1BB cell line highly expressing human 4-1BB;
FIGS. 3-(A)-(H): In vitro binding of 4-1BB antibody in CHO-K1-cynomolgus 4-1BB cell line highly expressing cynomolgus 4-1BB.
As shown in FIGS. 2-(A)-(I), all of the anti-4-1BB antibodies of the present invention can bind to human 4-1BB, and the detected antibody-binding ability is positively correlated with the antibody concentration. Increase with correlation. These antibodies are able to more sensitively bind human 4-1BB at lower concentrations compared to the reference antibodies (Utomilumab and Utomilumab) and correspond to the EC50 of the reference antibodies Utomilumab and Utomilumab.
As shown in FIGS. 3-(A)-(H), all of the anti-4-1BB antibodies of the present invention can bind to monkey 4-1BB, and the detected antibody-binding ability is positive with the antibody concentration. increases with a correlation of These antibodies are able to more sensitively bind to monkey 4-1BB at lower concentrations compared to the reference antibody Tab (Utomilumab) and are comparable or superior to the EC50 of the reference antibody Utomilumab. On the other hand, the reference antibody urelumab has no cross-binding activity with monkey 4-1BB.

3.4 Antigen binding proteins can activate the 4-1BB pathway in vitro CHO-K1 cells overexpressing human CD32b (CHO-K1/CD32b, Genscript , #M00600) was treated at 37°C for 30 minutes. After that, the cells were washed four times with F-12K culture medium containing 10% FBS. The treated cells were placed in a 96-well plate at 1.5×10 ⁴ per well and cultured overnight in a 37° C. incubator. The following day, human CD3-positive T cells were isolated from human PBMC using MACS kit (Miltenyi Biotec, #130-096-535). First, the number of cells was determined, then corresponding amounts of MACS buffer and Pan-T cell biotin antibody were added according to the number of cells, mixed well, and allowed to stand at 4°C for 5 minutes. Then, the corresponding amount of micro magnetic beads was added and allowed to stand at 4°C for 10 minutes. CD3-positive T cells passed through the LS column. The culture medium of the 96-well plate of the previous day was washed away, and 1×10 ⁵ purified T cells were added per well. Corresponding concentrations of 4-1BB antibody or control antibody were then added and OKT3 (eBiosciences, #16-0037-85) was added to a final concentration of 0.3 μg/ml. It was cultured for 72 hours in a 37°C insulated box. After 72 hours, supernatants were harvested and IFN-γ content was detected using an ELISA kit (Invitrogen, #88-7316-88). Add coating antibody to 96 flat bottom plate and overnight at 4°C. Next day, add ELISA buffer for 1 hour at room temperature. The recovered supernatant was added and cultured at room temperature for 2 hours. Wash plate twice and add detection antibody for 1 hour at room temperature. Plates were washed twice, HRP-streptavidin was added and incubated for 1 hour at room temperature. TMB substrate was then added, followed by ELISA stop solution (BBI, #E661006-0200). Absorbance values (OD ₄₅₀ to OD 570 ) at 450 nm and ₅₇₀ nm were read with a microplate reader (PerkinElemer, #Enspire) to calculate the concentration of IFN-γ.

The results are shown in FIGS. 4-(A)-(E): The 4-1BB antibody has the function of activating the 4-1BB pathway and inducing the activation of T cells. PR001758, PR001760, PR001764, PR001771, PR001774, PR001780, PR001781, PR001830, PR001833, PR001836, PR001838, PR001840, and PR000448 are all stronger than utomilumab , showed that the signal of IFN-γ was higher than that of utomilumab .

Example 4. Preparation of B7-H4×4-1BB Bispecific Antibodies Anti-B7-H4 antibodies selected from Example 2 and anti-4-1BB antibodies selected from Example 3 are combined to generate bispecific antibodies. 2, one end of which can identify B7-H4 specifically expressed on the tumor cell surface and the other end of which can bind the 4-1BB molecule on T cells. Targets can be bound simultaneously. After the B7-H4x4-1BB biantibody molecule binds to the tumor cell surface, it can recruit and activate T cells in the vicinity of the tumor cell to kill the tumor cell.
The B7-H4x4-1BB bispecific antibody generated in this example comprises multiple molecular structures:
FIGS. 7-(A)-(I) are structural schematic diagrams of the bispecific molecules of the present invention.

4.1 Construction of IgG-scFv tetravalent symmetric bispecific antibody Bispecific antibody of IgG-scFv tetravalent symmetric structure using anti-B7-H4 H2L2 antibody and anti-4-1BB H2L2 antibody built. The binding protein of the IgG-scFv tetravalent symmetric structure (as shown in FIG. 5-(A)) comprises two polypeptide chains: a polypeptide chain, also called a short chain, comprising VL_A-CL from the amino terminus to the carboxy terminus; Peptide chain 1; Polypeptide chain 2, also called long chain, comprising VH_A-CH1-h-CH2-CH3-L1-VH_BL2-VL_B from amino-terminus to carboxyl-terminus. h is the hinge region or derived sequence of an IgG antibody. Connecting peptide L1 and connecting peptide L2 of polypeptide chain 2 may be the sequences listed in Table 8.

4.2 Construction of IgG-VH tetravalent symmetric bispecific antibody Bispecific antibody of IgG-VH tetravalent symmetric structure using anti-B7-H4 H2L2 antibody and anti-4-1BB HCAb antibody built. A binding protein of IgG-VH tetravalent symmetric structure (as shown in FIG. 5-(B)) comprises two polypeptide chains: a polypeptide chain, also called a short chain, comprising VL_A-CL from amino-terminus to carboxy-terminus; Peptide chain 1; Polypeptide chain 2, also called long chain, comprising VH_A-CH1-h-CH2-CH3-L-VH_B from amino-terminus to carboxyl-terminus. h is the hinge region or derived sequence of an IgG antibody. In one embodiment, CH3 of polypeptide chain 2 is directly fused to VH_B, ie, the length of L is 0. In another embodiment, CH3 of polypeptide chain 2 is linked to VH_B via connecting peptide L;

4.3 Construction of IgG-VH-VH Hexavalent Symmetrical Bispecific Antibody A bispecific antibody was constructed. A binding protein of IgG-VH-VH hexavalent symmetric structure (as shown in FIG. 5-(C)) comprises two polypeptide chains: also called short chains, VL_A-CL from the amino terminus to the carboxy terminus. polypeptide chain 1 comprising; polypeptide chain 2, also called long chain, comprising VH_A-CH1-h-CH2-CH3-L1-VH_BL2-VH_B from amino-terminus to carboxyl-terminus; h is the hinge region or derived sequence of an IgG antibody. In one embodiment, CH3 of polypeptide chain 2 is directly fused to VH_B, ie, the length of L is 0. In another embodiment, the connecting peptides L1 and L2 of polypeptide chain 2 may be the sequences listed in Table 8.

4.4 Construct a bispecific antibody molecule with Fab-HCAb structure Construct a bispecific antibody with Fab-HCAb symmetric structure using anti-B7-H4 H2L2 antibody and anti-4-1BB HCAb antibody, It includes two different structures of Fab(CL)-VH-Fc (as shown in FIG. 5-(D)) and Fab(CH)-VH-Fc (as shown in FIG. 5-(E)). The binding protein of Fab(CL)-VH-Fc comprises two polypeptide chains: polypeptide chain 1, also called the short chain, comprising VH_A-CH1 from the amino terminus to the carboxy terminus; Polypeptide chain 2 comprising VL_A-CL-L1-VH_BL2-CH2-CH3 from base terminus to carboxyl terminus. The connecting peptides L1 and L2 of polypeptide chain 2 may be the sequences listed in Table 8. The binding protein of Fab(CH)-VH-Fc comprises two polypeptide chains: polypeptide chain 1, also called the short chain, comprising VL_A-CL from the amino terminus to the carboxy terminus; Polypeptide chain 2 comprising VH_A-CH1-L1-VH_B-L2-CH2-CH3 from base terminus to carboxyl terminus. The connecting peptides L1 and L2 of polypeptide chain 2 may be the sequences listed in Table 8.

4.5 Fab-Fc-VH(N) bispecific antibody of asymmetric structure using anti-B7-H4 H2L2 antibody and anti-4-1BB HCAb antibody to construct asymmetric structure built. Binding proteins of the Fab-Fc-VH(N) asymmetric structure (as shown in FIGS. 5-(F)(G)(H)) contain three polypeptide chains: Polypeptide chain 1 comprising VL_A-CL to the carboxy terminus; Polypeptide chain 2, also called long chain, comprising VH_A-CH1-h-CH2-CH3 from the amino terminus to the carboxy terminus; from the amino terminus to the carboxy terminus. Polypeptide chain 3 comprising VH_B-h-CH2-CH3, or polypeptide chain 3 comprising VH_BL-VH_B-h-CH2-CH3 from amino terminal to carboxy terminal, or polypeptide chain 3 comprising amino terminal to carboxy terminal Polypeptide chain 3 comprising VH_BL1-VH_BL2-VH_Bh-CH2-CH3. h is the hinge region or derived sequence of an IgG antibody. The connecting peptides L1 and L2 of polypeptide chain 3 may be the sequences listed in Table 8.

Formation of side products of mismatched heavy chains (e.g., mismatches of the two heavy chains of anti-4-1BB antibody or the two heavy chains of anti-B7-H4), as described in WO2009080251 and WO2009080252 Mutated heterodimeric Fc regions with “knob-hole” mutations and engineered disulfide bonds were used to minimize .

4.6 Construction of Fab-VH-Fc-VH symmetrical structure Construction of bispecific antibody with Fab-VH-Fc-VH symmetrical structure using anti-B7-H4 H2L2 antibody and anti-4-1BB HCAb antibody bottom. A binding protein of Fab-VH-Fc-VH symmetrical structure (as shown in FIG. 5-(I)) comprises two polypeptide chains: also called short chains, VL_A-CL from the amino terminus to the carboxy terminus. polypeptide chain 1 comprising; polypeptide chain 2, also called long chain, comprising VH_A-CH1-L1-VH_B-CH1-h-CH2-CH3-VHB from amino-terminus to carboxyl-terminus; h is the hinge region or derived sequence of an IgG antibody. The connecting peptides L1 and L2 of polypeptide chain 3 may be the sequences listed in Table 8. PR002408 (H: gm) related to PR007379 in Table 11-(B) is the same as the CDR of PR002408, and FR2 of the variable region VH has two mutations, the two mutations being T69I, It is E101G.

The amino acid sequences of the polypeptide chains of the double antibody molecules obtained according to the invention are shown in the sequence listing below.

The CDR sequence numbers for the antigen domains of the B7-H4x4-1BB bispecific antibody are shown in the table below, in Table 10, the 1# number is the antigen domain that binds B7-H4 and the 2# number is It is the antigen domain that binds 4-1BB.

Tables 11-(A)-(F) are information on the bispecific antibody molecular structure of the present invention.

Tables 12-(A)-(F) are the protein expression and physicochemical properties of the bispecific antibodies of the invention.

Example 5. FACS detection of in vitro binding of B7-H4x4-1BB bispecific antibodies in CHO-K1 cell lines overexpressing human and cynomolgus monkey 4-1BB. To study the in vitro binding activity of the 4-1BB arm of the antibody to human and cynomolgus monkey 4-1BB. A CHO-K1 cell line overexpressing human 4-1BB (CHO-K1/hu 4-1BB, Genscript, #M00538) and a CHO-K1 cell line overexpressing cynomolgus monkey 4-1BB (CHO-K1/cyno 4- 1BB, Genscript, #M00569) were used to perform antibody binding experiments at the cellular level. Briefly, cells CHO-K1/hu 4-1BB and CHO-K1/cyno 4-1BB cells were digested, resuspended in F12K complete medium and washed once with PBS. The cell density was adjusted to 1×10 ⁶ cells/ml each with PBS. After seeding 96-well V-bottom plates (Corning, #3894) with 100 μL cells/well, 100 μL/well of antibody to be measured diluted in a 3-fold gradient of 2-fold the final concentration was added. Cells were placed at 4°C and incubated for 1 hour away from light. Cells were then washed twice by adding 100 μL/well pre-chilled PBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Additionally 100 μL/well fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, #: 109-545-06, diluted 1:500) or (Ale xa ^Fluor® 647, Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, #: 109-605-098, 1:1000 dilution) and incubated at 4°C for 30 minutes away from light. Cells were washed twice with 100 μL/well pre-chilled PBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended with 200 μL/well of pre-chilled PBS and fluorescence emission signal values were read using a BD FACS CANTOII or ACEA NovoCyte series flow cytometer. .

FIG. 6-(A)-(L): In vitro binding of B7-H4×4-1BB bispecific antibody in CHO-K1 cell line overexpressing human 4-1BB;
FIG. 7-(A)-(C): In vitro binding of B7-H4×4-1BB bispecific antibody in CHO-K1 cell line overexpressing cynomolgus monkey 4-1BB;
As shown in FIG. 6-(A)-(L), the B7-H4×4-1BB bispecific antibodies in this example are both specific to CHO-K1 cells overexpressing human 4-1BB. Combined. PR002789, PR002972, PR003334, PR003335, PR003336, PR003337, PR003338, PR004160, PR004161, PR004158, PR004162, PR004357, PR004359, PR004279, PR004280, PR Binding of 004281, PR004282, PR005829, etc. to human 4-1BB is strong. Specifically, the Span value of the binding curve for antibody PR003335 is approximately 1.5-fold that of urelumab, and the MFI maximum is higher than that of urelumab.
As shown in FIGS. 7-(A)-(C), the B7-H4×4-1BB bispecific antibodies in this example are both specific to CHO-K1 cells overexpressing monkey 4-1BB. Combined. On the other hand, the reference antibody urelumab has no cross-binding activity with monkey 4-1BB.

Example 6. FACS detection of in vitro binding of the B7-H4x4-1BB bispecific antibody in a SK-BR-3 cell line that highly expresses B7-H4. to study the binding activity of the B7-H4 arm of B7-H4 to human B7-H4. A cell-level binding experiment with human B7-H4 was performed using SK-BR-3 cell line that highly expresses B7-H4. Briefly, SK-BR-3 cell suspensions were collected and cell densities were adjusted to 2×10 ⁶ cells/mL respectively. 96-well V-bottom plates (Corning, #: 3894) were seeded with 50 μL cells/well, followed by the addition of 50 μL/well of antibody to be measured diluted in a 3-fold gradient of 2-fold the final concentration. Cells were placed at 4°C and incubated away from light for 2 hours. Cells were then washed twice by adding 100 μL/well pre-chilled PBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. 100 μL/well fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, #: 109-605-098, 1:1000 dilution), 4 °C, light Incubate for 1 hour avoiding. Cells were washed twice with 100 μL/well of pre-chilled PBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended in 200 μL/well pre-chilled FACS and fluorescence emission signal values were read using an ACEA Novocyte 3000.

FIG. 8-(A)-(K): In vitro binding of B7-H4×4-1BB bispecific antibody in SK-BR-3 cell line highly expressing B7-H4;
As shown in FIGS. 8-(A)-(K), in this example, both B7-H4×4-1BB bispecific antibodies were specific for human B7-H4 on the SK-BR-3 cell line. binding efficaciously, with many binding curve EC50s between 0.1-10 nM.

Example 7. T cell activation experiments and cytokine release in the presence of the cell line SK-BR-3, which highly expresses the B7-H4 of the B7-H4x4-1BB bispecific antibody. To study the ability of bispecific antibodies to mediate target T cell activation in vitro, human pan T cells were used as effector cells and the cell line SK-BR-3, which highly expresses B7-H4, was In vitro activation experiments and detection of cytokine release were performed as cells mediating cross-linking. Specifically, 96-well flat-bottom plates (Corning, #3599) were coated with OKT3, the density of pan T was adjusted to 2×10 ⁶ cells/mL, and the density of SK-BR-3 was adjusted to 2×10 ⁵ cells/mL. /mL and the two cell suspensions were seeded into 96-well flat-bottom plates (Corning, #3599) at 50 μL cells/well each, followed by different gradient dilutions of 3-fold the final concentration. was added at 50 μL/well, in which the highest final antibody concentration was 100 nM or 30 nM or 20 nM or 6 nM, with a total of 6 or 3 or 2 concentrations of each antibody, and the final effective target ratio (effector cells to target cells (ie, effector cells:target cells) was 10:1 and two replicates were set up. At the same time, a homotypic IgG control group was set up in the plate. The 96-well plates were incubated in a carbon dioxide incubation chamber at 37°C for 2 or 3 days. After incubation was complete, the supernatant was removed, added to a 96-well plate (Corning, #3599) and centrifuged at 500g, 4°C for 5 minutes. Supernatants incubated for 2 days are used to detect the release of the cytokine IL-2, or supernatants incubated for 3 days are used to detect the release of IFN-gamma. The ELISA detection method is IL-2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88) kit and IFN gamma (IFN gamma Human Uncoated ELISA Kit, Thermo, #88-7316-88). refer

FIG. 9-(A)-(E): B7-H4×4-1BB bispecific antibody activates 4-1BB pathway in the presence of cell line SK-BR-3, which highly expresses B7-H4. induces and activates T cells to release IFN-γ.
FIG. 10-(A)-(N): B7-H4×4-1BB Bispecific Antibody B7-H4 High-Expressing Cell Line SK-BR-3 Activates 4-1BB Path in the Presence , induces and activates T cells to release IL-2.
As shown in FIGS. 9-(A)-(E) and FIGS. 10-(A)-(N), when a cell line SK-BR-3 highly expressing B7-H4 was present, B7-H4× 4-1BB Bispecific Antibody-Mediated T Cell Activation. and PR002790, PR002791, PR002802, PR002804, PR002806, PR003334, PR003335, PR003336, PR003337, PR003338, PR003487, PR003488, PR003489, PR004158, PR004160, PR004161, PR004162, PR004181, PR004182, PR004279, PR004280, PR004281, PR004282, PR004357, Activation of the 4-1BB path by PR004358, PR004359 is superior to or comparable to urelumab. Specifically, production of cytokines IFN-γ or IL-2 is higher than urelumab.
Different linker lengths also affect the activation of T cells by biantibodies, for example, PR004281 and PR004282 outperform PR003338 and urelumab (Fig. 10-K).
B7H4 arms with different antigenic epitopes affect T cell activation by dual antibodies, for example, PR004281 and PR004282 outperform PR004995 and Urelumab (Fig. 10-L).
Having different Fc mutation sites affects the activation of T cells by dual antibodies, eg PR005185, PR005186 and PR004282 are superior to urelumab (FIG. 10-M).

Example 8. Activation of the B7-H4x4-1BB bispecific antibody on T cells is independent of FcγR cells Study whether the activity of the B7-H4x4-1BB bispecific antibody depends on the expression of FcγRs To do so, in vitro activation of human pan T cells as effector cells and the cell line CHO-K1/CD32b, which highly expresses FcγRIIb, or the cell line CHO-K1/CD64, which highly expresses FcγMRI, as cells mediating cross-linking. Experiments and detection of cytokine release were performed. Specifically, 96-well flat bottom plates (Corning, #3599) were coated with OKT3, pan T density was adjusted to 2 x ¹⁰ cells/mL, and FcγR cell density was adjusted to 2 x ¹⁰ cells/mL. 96-well flat-bottom plates (Corning, #3599) at 50 μL cells/well each of the two cell suspensions, followed by 100 μL/well of the antibodies to be measured diluted in different gradients of 2-fold the final concentration. Two replicates were set up at a final effective target ratio of 10:1, added in wells. At the same time, a homotypic IgG control group was set up in the plate. The 96-well plates were incubated in a 37°C carbon dioxide incubation chamber for 2 days. After incubation was complete, 100 μl of supernatant was harvested, added to a 96-well plate (Corning, #3894), centrifuged at 500 g, 4° C. for 5 minutes, and supernatant harvested to detect cytokine IL-2 release. . For the ELISA detection method, refer to the operating instructions of the IL-2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88) kit.

Figures 11-(A)-(B): The specificity of the B7-H4x4-1BB double antibody for T cell activation is independent of FcγR expression. (A) T cell activation mediated by B7-H4x4-1BB bispecific antibody PR004282 is comparable to homotypic IgG control in the presence of cell line CHO-K1/CD32b cells that highly express FcγRIIb. Met. (B) T cell activation mediated by B7-H4x4-1BB bispecific antibody PR004282 is comparable to homotypic IgG control in the presence of cell line CHO-K1/CD64 cells that highly express FcγMRI. Met.

Example 9. Activation of B7-H4x4-1BB bispecific antibody against T cells depends on expression of B7-H4 Does B7-H4x4-1BB bispecific antibody activity depend on B7-H4 expression? To study whether human pan T cells were used as effector cells, the cell lines SK-BR-3 and MDA-MB-468, which highly express B7-H4, and COV644 and JIMT-1, which do not express B7-H4. and MDA-MB-231 cells as cells mediating cross-linking, in vitro activation experiments and detection of cytokine release. Specifically, 96-well flat-bottom plates (Corning, #3599) were coated with OKT3, pan T density was adjusted to 2 x ¹⁰⁶ cells/mL, and tumor cell density was adjusted to 2 x ¹⁰⁵ cells/mL. and seeding each of the two cell suspensions at 50 μL cells/well into a 96-well flat-bottom plate (Corning, #3599), followed by the addition of 50 μL/well of the antibody to be measured at 3× the final concentration, The final effective target ratio was 10:1 and two replicates were set up. At the same time, a homotypic IgG control group was set up in the plate. The 96-well plates were incubated in a 37°C carbon dioxide incubation chamber for 2 days. After incubation was complete, 100 μl of supernatant was harvested, added to a 96-well plate (Corning, #3894), centrifuged at 500 g, 4° C. for 5 minutes, supernatant harvested, and cytokine IL-2 released. For the ELISA detection method for detection, refer to the IL-2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88) kit operating instructions.

Figure 12: FACS detects B7-H4 expression levels on the surface of tumor cells. In FIG. 12-(A), the B7-H4 positive control antibody 1 was used to detect the B7-H4 expression level on the tumor cell surface. In FIG. 12-(B), the B7-H4 expression level on the tumor cell surface was detected using the B7-H4×4-1BB bispecific antibody. As shown, SK-BR-3 and MDA-MB-468 are tumor cells that highly express B7-H4, and JIMT-1, COV644 and MDA-MB-231 are tumor cells that do not express B7-H4. be.
Figures 13-(A)-(H): Activation of the B7-H4x4-1BB bispecific antibody against T cells specifically depends on the expression of B7-H4. (A), (B), in the presence of cell lines SK-BR-3 or MDA-MB-468 cells that highly express B7-H4, by the B7-H4×4-1BB bispecific antibody PR003334. Mediated T cell activation is superior to positive controls. In (C), (D), the extent of T cell activation mediated by PR003334 is comparable to homotypic IgG controls when cell lines COV644 or JIMT-1 cells that do not express B7-H4 are present. (E), (F) T cell activation mediated by B7-H4×4-1BB bispecific antibody PR004282 in the presence of cell line SK-BR-3 cells that highly express B7-H4. conversion is superior to the positive control urelumab. In (G), (H), the extent of T cell activation mediated by PR004282 is higher than that of homotypic IgG controls when cell lines JIMT-1 or MDA-MB-231 cells that do not express B7-H4 are present. are equivalent.

Example 10. Serum Stability Study of B7-H4x4-1BB Bispecific Antibody PR0003334 and PR000335 were used to study the stability of the B7-H4x4-1BB bispecific antibody in high concentration human serum. % human serum with an initial concentration of 100 nM and diluted to 8 concentration points with a 1:3 gradient. The samples were divided into 6 samples, incubated at 37°C for 0 days, 1 day, 2 days, 4 days, 7 days and 14 days, then quickly frozen in liquid nitrogen and left at -70°C for storage. SK-BR-3 cell line and CHO-K1-hu 4-1BB cells highly expressing B7-H4 after exposure of B7-H4×4-1BB bispecific antibody to concentrated serum at 37° C. for different times. was detected by FACS, see Example 3.3 and Example 6.

PR0004282 was gradient diluted with 95% human serum, divided into 5 tubes, incubated at 37°C for 0 days, 1 day, 2 days, 4 days, 7 days, 14 days, and then snap frozen in liquid nitrogen. Stored at -80 degrees. PR004282 after exposure to high concentration human serum at 37°C for different times detected in vitro binding to B7H4+ and 4-1BB+ cells using FACS method, the process is as follows:
CHO-K1/h4-1BB, CHO-K1/cyno4-1BB, CHO-K1/hB7H4 and CHO-K1/cynoB7H4 cells were digested, resuspended in complete medium with DMEM and washed with PBS before cell density was reduced to Each was adjusted to 1×10 ⁶ cells/mL. 96-well V-bottom plates (Corning, #3894) were seeded with 100 μL cells/well followed by a 3-fold gradient of 2-fold final concentration of target human serum-treated PR004282 antibody (for each antibody with different treatment times). dose containing 4.5% human serum) was added at 100 μL/well. Cells were placed at 4°C and incubated away from light for 2 hours. Furthermore, 100 μL/well of prechilled 2% FBS-containing PBS was added to wash the cells twice, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Additionally 100 μL/well of fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-06, diluted 1:1000) at 4° C. away from light. Incubated for 1 hour. Cells were washed twice with 100 μL/well pre-chilled PBS containing 2% FBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended at 200 μL/well in pre-chilled PBS containing 2% FBS and fluorescence emission signal values were read using an ACEA NovoCyte series flow cytometer.

Figure 14-(AD): Serum stability of B7-H4x4-1BB double antibodies PR003334 and PR003335. At different human serum incubation times, PR003334 and PR003335 showed no change in binding to SK-BR-3 cells and CHO-K1-human 4-1BB cells, which highly express B7-H4, in 90% serum. PR003335 was shown to have good serum stability.
FIG. 14-(EH): Serum stability of B7-H4×4-1BB double antibody PR004282. At different serum incubation times, PR004282 had no change in binding to CHO-K1 cells highly expressing B7H4 or 4-1BB in 95% serum, indicating that PR004282 has good serum stability. .

Example 11. FACS detection of the binding ability of the B7-H4x4-1BB bispecific antibody PR004282 at the 4-1BB+ cell level. To study in vitro binding activity to 4-1BB. CHO-K1 cell line overexpressing human 4-1BB (CHO-K1/h4-1BB, Genscript, #M00538), cynomolgus monkey 4-1BB CHO-K1 cell line (CHO-K1/cyno4-1BB, Genscript, # M00569), and a CHO-K1 cell line overexpressing mouse 4-1BB (CHO-K1/m4-1BB, Genscript, #M00568) was used to perform antibody binding experiments at the cellular level. Briefly, CHO-K1/h4-1BB cells, CHO-K1/cyno 4-1BB cells and CHO-K1/m4-1BB cells were digested and resuspended in PBS containing 2% FBS. Cell densities were adjusted to 1×10 ⁶ cells/mL respectively. After seeding 96-well V-bottom plates (Corning, #3894) with 100 μL cells/well, 100 μL/well of antibody to be measured diluted in a 3-fold gradient of 2-fold the final concentration was added. Cells were placed at 4°C and incubated away from light for 2 hours. Cells were then washed twice by adding 100 μL/well of prechilled FACS buffer containing 2% FBS in PBS, centrifuged at 500 g for 5 min at 4° C., and discarded the supernatant. In addition, 100 μL/well of fluorescent secondary antibody (Alexa ^Fluor® 647, Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, #109-605-098, 1:1000 dilution) was added at 4° C. with light. was avoided and incubated for 1 hour. Cells were washed twice with 100 μL/well of pre-chilled FACS buffer, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended using 200 μL/well pre-chilled FACS buffer and fluorescence emission signal values were read using an ACEA Novocyte 3000 flow cytometer.

As shown in FIG. 15-(A): in vitro binding status of B7-H4/4-1BB bispecific antibody PR004282 in CHO-K1 cell line highly expressing human 4-1BB; The binding activity of the h4-1BB cell line is superior to the control antibody Urelumab.
As shown in FIG. 15-(B): In vitro binding situation in CHO-K1 cell line highly expressing cynomolgus monkey 4-1BB of PR004282; No cross-reactivity.
As shown in FIG. 15-(C): In vitro binding situation in CHO-K1 cell line highly expressing mouse 4-1BB of PR004282; PR004282 has no mouse 4-1BB binding ability.

Example 12. FACS detection of the binding ability of B7-H4x4-1BB bispecific antibody PR004282 at the B7H4+ cell level. To study in vitro binding activity to H4. CHO-K1 cell line overexpressing human B7H4 (CHO-K1/hB7-H4, manufactured by HARBOUR BIOMED) Cynomolgus monkey B7-H4 CHO-K1 cell line (CHO-K1/cynoB7-H4, manufactured by HARBOUR BIOMED), mouse B7 -H4-overexpressing CHO-K1 cell line (CHO-K1/mB7-H4, manufactured by HARBOUR BIOMED) was used to perform antibody binding experiments at the cellular level. Briefly, CHO-K1/hB7H4, CHO-K1/cyno B7-H4 and CHO-K1/mB7-H4 cells were digested and resuspended in PBS containing 2% FBS. Cell densities were adjusted to 1×10 ⁶ cells/mL respectively. After seeding 96-well V-bottom plates (Corning, #3894) with 100 μL cells/well, 100 μL/well of antibody to be measured diluted in a 3-fold gradient of 2-fold the final concentration was added. Cells were placed at 4°C and incubated away from light for 2 hours. Cells were then washed twice by adding 100 μL/well of pre-chilled PBS containing 2% FBS, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded. An additional 100 μL/well of fluorescent secondary antibody (Alexa ^Fluor® 647, Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, #109-605-098, 1:1000 dilution) was added and incubated at 4°C. , and incubated for 1 hour away from light. Cells were washed twice with 100 μL/well pre-chilled PBS containing 2% FBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Finally, 200 μL/well of pre-chilled 2% BSA-containing PBS cells were resuspended and fluorescence emission signal values were read using an ACEA Novocyte 3000 flow cytometer.

As shown in FIG. 16-(A): In vitro binding of B7-H4/4-1BB bispecific antibody PR004282 in CHO-K1 cell line highly expressing human B7-H4; PR004282 and CHO-K1/hB7H4 cells. The binding activity of the system is higher than the B7H4 monoclonal antibody PR002408.
As shown in FIG. 16-(B): In vitro binding of B7-H4 antibody in CHO-K1 cell line highly expressing cynomolgus monkey B7-H4; Comparable binding activity.
As shown in FIG. 16-(C): In vitro binding of B7-H4 antibody in CHO-K1 cell line highly expressing mouse B7-H4; PR004282 has weak binding ability to mouse B7H4.

Example 13. FACS detection of the ability of the B7-H4x4-1BB bispecific antibody PR004282 to bind simultaneously to CHO-K1/h4-1BB and SK-BR-3. to study the ability of sex antibody PR004282 to bind simultaneously to CHO-K1/h4-1BB and SK-BR-3. Briefly, CHO-K1/h4-1BB cells and SK-BR-3 cells were digested and the cell density was adjusted to 1×10 ⁶ cells/mL each using PBS, and the cells were purified against SK-BR-3. CHO-K1/h4-1BB cells were stained with 0.5 μM Far-red (lifetechnologies, #C34572) and 0.5 μM CFSE (lifetechnologies, #C34544) for 5 minutes at room temperature. Centrifugation-stained cells were washed once with >20 ml medium containing 1% FBS. The washed cells were resuspended in FACS buffer and the cell density was adjusted to 2×10 ⁶ /ml. 50 μl SK-BR-3 cells (1×10 ⁵ cells per well) and 25 μl CHO-K1/h4-1BB (cells per well) were added per well in 96-well V-plates (Corning, #3894). 1×10 ⁵ ) and antibody to be measured diluted in 25 ul 4-fold gradient FACS buffer were added and mixed thoroughly. Incubated for 1 hour at 4°C. FITC+CHO-K1/h4-1BB cells and Alexa 647+SK-BR-3 double positive percentages were distinguished using an ACEA Novocyte 3000 flow cytometer. Data were analyzed using FlowJo software (Tree Star, Ashland, Oreg.) and the percentage of double-stained cells was used to determine the double antibody-mediated co-binding rate=(Q2/(Q1+Q2). .

FIG. 17 shows that only PR004282 has the ability to simultaneously bind CHO-K1/h4-1BB and SK-BR-3. None of the monoclonal antibodies are capable of this.

Example 14. FACS detection of the binding capacity of the B7-H4x4-1BB bispecific antibody PR004282 at the primary cell level. / to study the activity of cynomolgus monkey primary T cells. Monkey CD3 T cells were isolated from cryopreserved monkey PBMC (PharmaLegacy, #SC 1702051) using the Non-Human Primate CD3 Isolation Kit (Miltenyi, #130-092-012) and added to medium RPMI 1640 + 10% FBS + 1% sodium pyruvate. (Thermo, #1360-070) + 1% non-essential amino acids solution (Thermo, #11140-050) to resuspend to 2^106/ml, 1 ml each into a 6-well plate, 20 ng/ml PMA (Sigma, #P1585-1 mg) and 5 nM Ionomycin (Sigma, #407952-5 mg) was added for 16 h in a 37° C. CO ₂ incubation chamber. Human CD3+ T cells were isolated from cryopreserved human PBMCs (Saily, XFB-HP100B) using a human CD3 isolation kit (Miltenyi, #130-096-535), and other manipulations were the activation and activation of monkey CD3+ T cells. was the same.

Post-activation monkey T cell density was adjusted to 1×10 ⁶ cells/mL with PBS containing 2% FBS and seeded at 100 μL LCD3+ T cells/well in 96-well V-plates (Corning, #3894) followed by 100 μL/ml. Target antibody PR004282/hIgG1/Urelumab/hIgG4 diluted in a 3-fold gradient of 2-fold the final well concentration was added and the cells were placed at 4° C. and incubated for 2 hours protected from light. After that, 100 μL/well of pre-chilled 2% FBS in PBS was added, cells were washed twice, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. In addition, 100 μL/well of fluorescent secondary antibody (Alexa ^Fluor® 647, Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, #109-605-098, 1:1000 dilution) was added at 4° C. with light. was avoided and incubated for 1 hour. Cells were washed twice with 100 μL/well pre-chilled PBS containing 2% FBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended at 100 μL/well with pre-chilled PBS containing 2% FBS and fluorescence emission signal values were read using an ACEA Novocyte 3000 flow cytometer.

Zombie NIR ^™ dye (Biolegend, #77184) and activated human T cells were incubated at room temperature in the dark for 15-30 minutes, cells were washed with FACS buffer and cell density was adjusted to 1×10 ⁶ respectively. Adjusted to cells/mL. After inoculating 96-well V-bottom plates (Corning, #3894) at 100 μL LCD3+ T cells/well, 100 μL/well of the antibody to be measured PR004282/hIgG1 diluted in a 3-fold gradient of 2-fold the final concentration was added, and the cells were plated 4 times. °C and incubated away from light for 2 hours. Cells were then washed twice with 100 μL/well of pre-chilled PBS containing 2% FBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. In addition, 100 μL/well of fluorescent secondary antibody (Alexa ^Fluor® 488, Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, #109-545-098, 1:1000 dilution) was added at 4° C. with light. was avoided and incubated for 1 hour. Cells were washed twice with 100 μL/well pre-chilled PBS containing 2% FBS, centrifuged at 500 g, 4° C. for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended at 100 μL/well in pre-chilled PBS containing 2% FBS and fluorescence signal values were read using an ACEA Novocyte 3000 flow cytometer.

As shown in Figure 18-(AB), the B7-H4/4-1BB bispecific antibody PR004282 can bind to activated human or monkey CD3+ T cells.

Example 15. ELISA detection of cross-reactivity between B7-H4x4-1BB bispecific antibody PR004282 and B7 family or other members of the TNFR family 15.1 B7-H4x4-1BB bispecific antibody PR004282 and B7 family ELISA detection of cross-reactivity with other members of the B7 family (Table 13) were each diluted in PBS to 1 μg/ml and added to 96-well plates (Corning, #9018), 100 μl per well, 4° C. C overnight. After discarding the liquid, the cells were washed three times with PBST buffer (pH 7.4, containing 0.05% tween-20), added with 250 μl of 2% BSA blocking solution, and incubated at 37° C. for 1 hour. Discard the blocking solution, wash three times with PBST buffer, dilute the antigen binding protein to be measured to three concentrations of 100 nM, 10 nM and 1 nM, add 100 μl to each well, and incubate at 37° C. for 1 hour. incubated. After washing three times with PBST buffer, a 5000-fold diluted goat anti-human HRP secondary antibody (Invitrogen, #A18805) was added and incubated at 37°C for 1 hour while avoiding light. After washing three times with PBST buffer, 100 μl/well of TMB (Biopanda, #TMB-S-003) was added and left at room temperature for approximately 30 minutes away from light. 50 μl/well of stop solution (BBI life sciences, #E 661006-0200) was added to each well to stop the reaction and 450 nm absorbance (OD450) values were measured with a microplate reader (PerkinElemer, #Enspire).

Figure 19-(A) shows that the antibody PR004282 of the invention does not cross-react with other member proteins of the B7 family.

15.2 ELISA detection of cross-reactivity of B7-H4x4-1BB bispecific antibody PR004282 with other members of the TNFR family of proteins (Table 14), each diluted to 1 μg/ml in PBS, Added to 96-well plates (Corning, #9018) and incubated overnight at 4°C, 100 µl per well. After discarding the liquid, the cells were washed three times with PBST buffer (pH 7.4, containing 0.05% tween-20), added with 250 μl of 2% BSA blocking solution, and incubated at 37° C. for 1 hour. Discard the blocking solution, wash three times with PBST buffer, dilute the antigen binding protein to be measured to three concentrations of 100 nM, 10 nM and 1 nM, add 100 μl to each well, and incubate at 37° C. for 1 hour. Incubated, homotypic antibody served as control. After washing three times with PBST buffer, a 5000-fold diluted goat anti-human HRP secondary antibody (Invitrogen, #A18805) was added and incubated at 37°C for 1 hour while avoiding light. After washing three times with PBST buffer, 100 μl/well of TMB (Biopanda, #TMB-S-003) was added and allowed to stand at room temperature for approximately 30 minutes away from light. 50 μl/well stop solution (BBI life sciences #E 661006-0200) was added to each well to stop the reaction and 450 nm absorbance (OD450) values were measured with a microplate reader (PerkinElemer #Envision).
FIG. 19-(B) shows that the antibody PR004282 of the invention does not cross-react with other member proteins of the TNFR family.

Example 16. Detection of ADCC effect of B7-H4x4-1BB bispecific antibody PR004282 16.1 This example is the detection of ADCC effect of PR004282 by ADCC reporter gene cell line Jurkat FcγRIIIa-V158/NFAT-Luc cells ( HARBOUR BIOMED) is used to detect the activity of PR004282 on the ADCC effect of CHO-K1/h4-1BB or SK-BR-3. CHO-K1/h4-1BB or SK-BR-3 were centrifuged at 300 g for 5 minutes and then resuspended with RPMI 1640 + 4% FBS serum medium. Cell density was adjusted to 6×10 ⁵ cells/ml and 50 μl cell suspension was added to each well of a 96-well plate and incubated overnight at 37°C. Jurkat FcγRIIIa-V158/NFAT-Luc cells were centrifuged at 400 g for 4 minutes and then resuspended using RPMI 1640+4% FBS serum medium. Cell density was adjusted to 3×10 ⁶ cells/ml and 50 μl cell suspension was added to each well of a 96-well plate. Antibodies were diluted in RPMI 1640 + 4% FBS medium, with a maximum final concentration of 100 nM, each antibody at a total of 4 concentrations, diluted 5-fold, set up in duplicate, and 50 μl antibody dilution in each well of a 96-well plate. was added. At the same time, a homotypic IgG control group and a blank medium control group were placed in the plate. Cells and antibodies were incubated at 37°C for 5 hours. The 96-well plate was left at room temperature for 30 minutes and 60 μl/well of room temperature One-Glo color developer (Promega, #E6110) was added. The samples were then incubated for 10 minutes at room temperature away from light. Luminscence readings were performed using PE Enspire. Fold = Luminscence reading of antibody wells/Luminscence reading of blank media control group, graphed with Prism 8 software. PR004469 and PR003369 (affinity matured mutant B7H4 monoclonal antibody, having ADCC-enhancing effect, heavy chain sequence shown in SEQ ID NO: 290, light chain sequence shown in SEQ ID NO: 291) were used as positive controls, and human Iso IgG1 (CrownBio, #C0001-4) antibody served as a negative control.

Figures 20-(A)-(B) show that PR004282 does not exhibit obvious ADCC activity against CHO-K1/h4-1BB and SK-BR-3 cells. On the other hand, the positive controls PR004469 and PR003369 can induce strong ADCC effects on CHO-K1/4-1BB or SK-BR-3, respectively, in a dose-dependent manner.

16.2 This example is to study the activity of the B7H4/4-1BB double antibody PR004282 in mediating NK cell killing of target cells through ADCC effects.
In this experiment, human NK was used as the effector cell, and the cell line SK-BR-3, which highly expresses B7H4, was used as the target cell. An ACEA RTCA instrument was used to detect the electrical conductivity of the target cells and reflect the killing efficiency. The 96-well plate E-plate was first equilibrated with 50 μl of complete medium. SK-BR-3 cells were digested, resuspended in RPM1640 complete medium containing 10% fetal bovine serum, diluted to 4×10 ⁵ /ml and plated at 50 μl/well in E-plate 96 plates, or 2×10 ^{4 .} /well and incubated overnight at 37°C. The next day, NKs were selected using the EasySep ^™ Human CD56 Positive Selection Kit (Stem cell, #17855) and 50 μl of fresh medium containing 1×10 ⁵ PBMCs were added to each well, followed by 50 μl. Diluted antibodies were added in a 4× gradient, with a maximum final antibody concentration of 100 nM, each antibody was set for a total of 4 concentrations, 5-fold dilutions, and 2 replicates. PR003369 was used as a positive control, and human Iso IgG1 (CrownBio, C0001-4) antibody was used as a negative control. The electrical conductivity of the target cells was measured in real time and the 24 hour data was used to calculate the target cell killing efficiency, target cell killing efficiency = (1 - sample/iso control) x 100%.

The results in FIG. 20-(C) show no apparent killing of PR004282 compared to PR003369. PR004282 was shown to have no ADCC activity against SK-BR-3 target cells.

Example 17. An Octet Red 96e Molecular Interaction Analyzer was used for detection of antibody affinity to human, mouse, and cynomolgus monkey Fcγ receptor proteins by the BLI method, and the experimental buffer was 1× kinetics buffer (ForteBio, #18). -1105).
The rotation speed of the instrument was set at 1000 rpm and FAB2G sensors (Fortebio, #18-5125) placed in a row were first equilibrated in test buffer for 10 minutes, after which the antibodies were captured on the FAB2G sensors and high After equilibrating in buffer for 120 s, the FAB2G sensor binds to the diluted Fcγ receptor protein in a 2-fold gradient, setting the binding time to 60 s and the dissociation time to 30 s. 15. Finally, the FAB2G sensor was regenerated by immersion in a 10 mM glycine-HCl pH 1.5 solution to elute protein bound to the sensor. Affinity tests between antibodies and FcRn were performed under two conditions, buffer pH 6.0 and 7.4, respectively. For cynomolgus monkey CD32b and CD64 with an avi tag, the SA sensor (Fortebio, 18-5019) was used to capture the receptor protein and the antibody was the analyte.

If data analysis is performed using Octet Data Analysis software (Fortebio, version 11.0), 0 nM is the reference well, reference subtraction is subtracted, and the "1:1 Global fitting" method is selected. Data fitting was performed and kinetic parameters of protein-antibody binding were calculated to obtain kon(1/Ms), kdis(1/s) and KD(M) values. For fast binding fast dissociating interactions, the "steady state" method was chosen to fit the data and obtain KD(M) values.

As can be seen from Tables 16-18, PR004282 has higher KD values than the counter-reference antibody urelumab, and binds weakly to the human mouse monkey Fcγ receptor protein.

Example 18. Detection of Non-Specific Cytokine Release of B7-H4x4-1BB Bispecific Antibody To assess whether the B7-H4x4-1BB bispecific antibody causes non-specific release of cytokines, Monocytes were isolated using a human CD14 isolation kit (Meltenyi, #130-050-201), or PBMCs from the same donor were resuspended in RPM1640 complete medium containing 10% fetal bovine serum and plated to 2E6/ml. Dilute and plate in 96-well plates at 100 μl/well (Costar, #3599), ie 2E5/well, followed by addition of 100 ul 2× final concentration gradient of diluted antibody, top concentration of antibody is 300 nM, 10-fold Dilutions were set up in duplicate for a total of 3 concentrations and incubated overnight at 37°C. Urelumab was used as a positive control, and Utomilumab and human Iso IgG1 (CrownBio, C0001-4) antibody were used as negative controls. After 24 h of incubation, supernatants were collected to detect the release of TNF-a (Thermo, 88-7066-88) and IL-6 (Thermo, 88-7346-77), and after 72 h of incubation, supernatants were collected. , the release of IFN-gamma (Thermo, 88-7316-77) was detected.

FIG. 21-(A)-(C): Cytokine release after co-incubation of B7-H4×4-1BB bispecific antibody PR004282 with PBMCs or monocytes. Whether PBMC or monocytes, urelumab showed strong TNF-a and IL-6 release, and IFN-gamma appeared only upon co-incubation with PBMC. On the other hand, PR004282 showed excellent safety similar to Utomilumab, demonstrating clear cytokine release regardless of incubation with PBMCs or monocytes.

Example 19. Pharmacokinetic Studies of B7-H4x4-1BB Bispecific Antibodies in Normal Mice This example examines the pharmacokinetic properties of the fusion proteins. The method was as follows: Six female C57BL/6 mice weighing 18-22 grams were selected and bispecific by intravenous injection at a dose of 5 mg/kg PR003334 or 5 mg/kg PR003335 or 5 mg/kg PR004282. Antibody drug was administered and 3 animals in one group had whole blood drawn before administration and at 15 minutes, 24 hours (1 day), 4 days and 10 days after administration, and another 3 animals received administration. Whole blood was collected before and 5 hours, days 2, 7, and 14 after dosing. Whole blood was left to clot for 30 minutes, then centrifuged at 2000 rpm for 5 minutes at 4°C and separated serum samples were stored frozen at -80°C until analysis. In this example, two ELISA methods were used to quantitatively measure drug concentrations in mouse serum. ELISA method 1, thus a global detection method, captures human Fc-containing fusion proteins in mouse sera by goat anti-human Fc polyclonal antibodies coated on 96-well plates, followed by HRP-labeled goat anti-human Fc secondary Add antibody for detection. ELISA method 2, a functional domain detection method, captures human 4-1BB HCAb-containing bispecific antibodies in mouse sera by human 4-1BB protein coated on 96-well plates, followed by HRP-labeled goat anti-antibodies. A human Fc secondary antibody is added for detection. A non-compartmental model (NCA) was selected to analyze blood concentration data and evaluate its pharmacokinetics using Phoenix WinNonlin software version 8.2.

Figures 22-(A)-(F) and Table 19 show pharmacokinetic data for B7-H4x4-1BB bispecific antibodies PR003334 and PR003335 and PR004282. The results in FIG. 22-(A) indicate that the half-life of PR003334 in mice is approximately 8 days in the overall detection method. FIG. 22-(B) The functional domain detection method indicates that the half-life of PR003334 in mice is about 9 days. In the overall method of Figure 22-(C), the half-life of PR003335 in mice was approximately 14 days. FIG. 22-(D) The functional domain detection method indicates that the half-life of PR003335 in mice is approximately 12 days. In the overall method of FIG. 22-(E), the half-life of PR004282 in mice was about 11 days. FIG. 22-(F) Functional domain detection method indicates that the half-life of PR004282 in mice is about 8 days.

Example 20. Pharmacokinetic Studies of B7H4/4-1BB Bispecific Antibody PR004282 in Cynomolgus Monkeys This example was conducted by examining the pharmacokinetic process of the B7-H4/4-1BB bispecific antibody in cynomolgus monkeys. Specifically, PR004282 was diluted in 0.9% saline and injected intravenously into male cynomolgus monkeys at a dose of 1 mg/kg and treated with 0.5, 1, 2, 4, 8, 0.5, 1, 2, 4, 8, 1 mg/kg doses pre- and post-dose. Blood samples were taken at 12, 24, 48, 72, 168, 336, 504, 672, 840, 1008 hours. Blood samples were allowed to stand at room temperature for 30 minutes and then centrifuged to obtain serum. The content of B7-H4/4-1BB bispecific antibody in serum was detected using an established specific ELISA method (global detection method).

As shown in Figure 23 and Table 20, the B7-H4/4-1BB bispecific antibody exhibited typical IgG-like pharmacokinetic curves after intravenous administration in cynomolgus monkeys. The half-life of PR004282 in male monkeys was approximately 3 days.

Example 21. Antitumor efficacy evaluation of B7-H4x4-1BB bispecific antibody in BALB/c-hCD137/CT26-B7-H4 mouse model In vivo antitumor efficacy of B7-H4x4-1BB bispecific antibody To evaluate , 6-8 week old female BALB/c-hCD137 mice were used, after which each experimental mouse was inoculated subcutaneously with 5×10 ⁵ CT26/hB7-H4 cells on the day of tumor cell inoculation. When the mean tumor volume reached 80 ^mm3 , mice were randomly grouped, 6 mice per group. After grouping, specific concentrations of drugs diluted in PBS were administered by intraperitoneal injection (ip) twice a week for a total of 6 doses (BIW*3), with PBS serving as blank control group. Tumor volumes and mouse body weights were measured on the day of the first dose and on days 4, 7, 11, 14 and 18. Tumor size calculation formula: tumor volume (mm ³ )=0.5×(tumor length×tumor width ² ).

Figure 24 showed the anti-tumor efficacy of the B7-H4x4-1BB bispecific antibody in the BALB/c-hCD137/CT26-B7-H4 mouse model. As shown in FIG. 24-(A), the B7-H4×4-1BB bispecific antibody PR003334 was efficacious in suppressing tumor growth in mice at 18 mpk and 6 mpk concentrations. As shown in FIG. 24-(B), changes in body weight of mice in each tested group were all within the normal range.

Example 22. Antitumor Efficacy Evaluation of B7-H4x4-1BB Bispecific Antibody in MDA-MB-468 Xenograft Mouse Model To evaluate in vivo antitumor efficacy of B7-H4x4-1BB bispecific antibody 6-8 week old female NCG mice were used, after which each experimental mouse was inoculated subcutaneously with 5×10 ⁶ MDA-MB-468 cells on the day of tumor cell inoculation. When the mean tumor volume reached 120 ^mm3 , the mice were randomly grouped, 6 mice per group. After grouping, human 5×10 ⁶ PBMC cells were inoculated, and the next day, drugs at specific concentrations diluted in PBS were injected intraperitoneally (ip) twice a week for a total of 6 times (BIW*3). The same type of control IgG was used as a control group. Tumor volumes and mouse body weights were measured on the day of the first dose and on days 7, 12, 15, 19, 22, 26 and 29. Tumor size calculation formula: tumor volume (mm ³ )=0.5×(tumor length×tumor width ² ).

Figure 25 showed the anti-tumor efficacy of the B7-H4x4-1BB bispecific antibody in the MDA-MB-468 xenograft mouse model. As shown in FIG. 25-(A), the B7-H4×4-1BB bispecific antibody PR003338 has efficacy in suppressing tumor growth in mice at a concentration of 18 mpk, and the efficacy of urelumab at a concentration of 15 mpk. Better than As shown in FIG. 25-(B), changes in body weight of mice in each tested group were all within the normal range.

Example 23. Antitumor efficacy evaluation of B7-H4x4-1BB bispecific antibody in OVCAR3 xenograft mouse model To evaluate the in vivo antitumor efficacy of the B7-H4x4-1BB bispecific antibody, 6- Eight-week-old female NCG mice were used, after which each experimental mouse was subcutaneously inoculated with 5×10 ⁶ OVCAR3 cells on the day of tumor cell inoculation. When the mean tumor volume reached 120 ^mm3 , the mice were randomly grouped, 6 mice per group. After grouping, human 5×10 ⁶ PBMC cells were inoculated, and the next day, drugs at specific concentrations diluted in PBS were injected intraperitoneally (ip) twice a week for a total of 6 times (BIW*3). The same type of control IgG was used as a control group. Tumor volumes and mouse body weights were measured on the day of the first dose and on days 7, 12, 15, 19, 22, 26 and 29. Tumor size calculation formula: tumor volume (mm ³ )=0.5×(tumor length×tumor width ² ).

Figure 26 showed the anti-tumor efficacy of the B7-H4x4-1BB bispecific antibody PR004282 in the OVCAR3 xenograft mouse model. As shown in FIG. 26-(A), the B7-H4×4-1BB bispecific antibody PR004282 was effective in suppressing tumor growth in mice at a concentration of 18 mpk. As shown in FIG. 26-(B), changes in body weight of mice in each tested group were all within the normal range.

Example 24. Evaluation of anti-tumor efficacy and memory immune effect of B7-H4×4-1BB bispecific antibody in BALB/c-hCD137/CT26-hB7-H4 mouse model To assess anti-tumor efficacy in vivo, 6-8 week old female BALB/c-hCD137 mice were used followed by 5×10 ⁵ CT26-B7-H4 cells in each experimental mouse on the day of tumor cell inoculation. inoculated subcutaneously. When the mean tumor volume reached 80 ^mm3 , mice were randomly grouped, 6 mice per group. After grouping, specific concentrations of drugs diluted in PBS were administered by intraperitoneal injection (i.p.) twice a week for a total of 6 times (BIW*3), with PBS serving as a blank control group and urelumab as a positive used as a control group. Tumor volume and mouse body weight measurements were taken on the day of the first dose and three times a week thereafter for 45 days. Tumor size calculation formula: tumor volume (mm ³ )=0.5×(tumor length×tumor width ² ). Forty-five days after administration, 5×10 ⁵ CT26/hB7H4 cells were subcutaneously inoculated on the contralateral side of the tumor-removed mice, and mice of the same age were used as a newly inoculated control group. Tumor volumes and mouse body weights were measured 3 times a week and mice were sacrificed on day 66.

Figure 27 showed the anti-tumor efficacy and memory immune efficacy of the B7-H4x4-1BB bispecific antibody PR004282 in the BALB/c-hCD137/CT26-B7-H4 mouse model. As shown in FIG. 27-(A)-(D), the B7-H4×4-1BB bispecific antibody PR004282 has efficacy in suppressing tumor growth in mice at a concentration of 1 mpk, equivalent to urelumab efficacy. is. As shown in FIG. 27-(E), changes in body weight of mice in each tested group were all within the normal range. As shown in FIG. 27-(F)-(I), after tumor removal mice were freshly inoculated with the same tumor, tumors no longer grew compared to uninoculated, untreated, age-matched mice, and PR004282 , have shown that the production of memory immune T cells can be induced and sustained effective tumor killing in the long term.

Example 25. Evaluation of Antitumor Efficacy Specificity of B7-H4x4-1BB Bispecific Antibody 25.1 Evaluation of Antitumor Efficacy Specificity of B7-H4x4-1BB Bispecific Antibody in JIMT-1 Xenograft Mouse Model Evaluation Six to eight week old female NCG mice were used, after which each experimental mouse was subcutaneously inoculated with 5×10 ⁶ JIMT-1 cells on the day of tumor cell inoculation. When the mean tumor volume reached 120 ^mm3 , the mice were randomly grouped, 6 mice per group. After grouping, human 5×10 ⁶ PBMC cells were inoculated, and the next day, drugs at specific concentrations diluted in PBS were injected intraperitoneally (ip) twice a week for a total of 6 times (BIW*3). The same type of control IgG was used as a control group. Tumor volumes and mouse body weights were measured on the day of the first dose and on days 7, 12, 15, 19, 22, 26 and 29. Tumor size calculation formula: Tumor volume (mm ³ )=0.5×(tumor major axis×tumor minor axis ² ).

FIG. 28-(A) showed the anti-tumor efficacy of PR004282 in the JIMT-1 xenograft mouse model. As shown in the figure, PR004282 has no efficacy in suppressing tumor growth in mice at a concentration of 18 mpk, Urelumab has a certain efficacy at a concentration of 15 mpk, JIMT-1 is a cell that does not express B7H4, and PR004282 , indicating the inability to kill tumor cells that do not express B7H4.

25.2 Evaluation of Antitumor Efficacy Specificity of B7-H4x4-1BB Bispecific Antibody by BALB/c-hCD137/CT26 Mouse Model To assess the specificity of tumor efficacy, 6-8 week old female BALB/c-hCD137 mice were used, after which each experimental mouse was subcutaneously inoculated with 5×10 ⁵ CT26 wild-type cells on the day of tumor cell inoculation. . When the mean tumor volume reached 80 ^mm3 , mice were randomly grouped, 6 mice per group. After grouping, specific concentrations of drugs diluted in PBS were administered by intraperitoneal injection (ip) twice a week for a total of 6 times (BIW*3), with PBS serving as a blank control group. Tumor volumes and mouse body weights were measured on the day of the first dose and on days 4, 7, 11, 14 and 18 thereafter. Tumor size calculation formula: Tumor volume (mm ³ )=0.5×(tumor major axis×tumor minor axis ² ).

FIG. 28-(B): B7-H4×4-1BB bispecific antibody PR004282 did not show strong tumor growth inhibitory efficacy against mice in the BALB/c-hCD137/CT26 mouse model, whereas urelumab It showed strong efficacy. CT26 is a cell that does not express B7H4, indicating that PR004282 cannot kill cells that do not express B7H4. On the other hand, according to the results of Example 24, growth of CT26-hB7-H4 tumor cells is inhibited by PR004282 after overexpressing human B7H4 on CT26 cells. Combining the two experiments, it can be inferred that tumor cell killing by PR004282 is dependent on B7H4 expression.

Although specific embodiments of the present invention have been described above, it should be understood that these are merely examples and that various changes and modifications can be made to these embodiments without departing from the principles and essence of the present invention. Those skilled in the art should understand. Therefore, the protection scope of the present invention is defined by the attached claims.

Claims (25)

A bispecific antibody comprising an antigen binding domain targeting B7-H4 and an antigen binding domain targeting 4-1BB. The antigen-binding domain targeting B7-H4 comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16 or 23, and HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 46, 59 or 60. wherein said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 98 or 84;
Preferably, said antigen binding domain targeting B7-H4 is HCDR1 as shown in SEQ ID NO: 16 as sequence, HCDR2 as shown in SEQ ID NO: 46 as sequence and HCDR3 as shown in SEQ ID NO: 84 as sequence; HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 59 as sequence and HCDR3 shown in SEQ ID NO: 98 as sequence; or HCDR1 shown in SEQ ID NO: 16 as sequence, HCDR2 shown in SEQ ID NO: 60 as sequence and preferably VH shown in SEQ ID NO: 142, VH shown in SEQ ID NO: 159 or VH shown in SEQ ID NO: 160 as sequence; more preferably heavy chain shown in SEQ ID NO: 174 as sequence , comprising the heavy chain set forth in SEQ ID NO: 191 or the heavy chain set forth in SEQ ID NO: 192;
More preferably, said antigen-binding domain targeting B7-H4 further comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of said LCDR1 is shown in SEQ ID NO: 112 or 113, and the amino acid sequence of said LCDR2 is shown in SEQ ID NO: 118; the LCDR3 amino acid sequence is shown in any of SEQ ID NOS: 131-133;
Even more preferably, said antigen binding domain targeting B7-H4 further comprises LCDR1 as shown in SEQ ID NO: 112 as sequence, LCDR2 as shown in SEQ ID NO: 118 as sequence and LCDR3 as shown in SEQ ID NO: 131 as sequence; LCDR1 shown in SEQ ID NO: 113, LCDR2 shown in SEQ ID NO: 118 as sequence and LCDR3 shown in SEQ ID NO: 132 as sequence; or LCDR1 shown in SEQ ID NO: 112 as sequence, LCDR2 shown in SEQ ID NO: 118 as sequence and LCDR3 as shown in SEQ ID NO: 133 as a sequence; preferably further comprises VL as shown in any of SEQ ID NOS: 166-169 as sequences; 2. The bispecific antibody of claim 1, comprising chains. The antigen-binding domain targeting B7-H4 includes HCDR1 shown in SEQ ID NO: 16 as a sequence, HCDR2 shown in SEQ ID NO: 46 as a sequence, HCDR3 shown in SEQ ID NO: 84 as a sequence, and HCDR3 shown in SEQ ID NO: 112 as a sequence. LCDR1 as shown, LCDR2 as shown in SEQ ID NO: 118 and LCDR3 as shown in SEQ ID NO: 131 as sequence; HCDR3 shown, LCDR1 shown in SEQ ID NO: 113 as sequence, LCDR2 shown in SEQ ID NO: 118 as sequence and LCDR3 shown in SEQ ID NO: 132 as sequence; or HCDR1 shown in SEQ ID NO: 16 as sequence, SEQ ID NO: HCDR2 shown in SEQ ID NO: 60 and HCDR3 shown in SEQ ID NO: 84 as sequence, LCDR1 shown in SEQ ID NO: 112 as sequence, LCDR2 shown in SEQ ID NO: 118 as sequence and LCDR3 shown in SEQ ID NO: 133 as sequence;
Preferably, said antigen binding domain targeting B7-H4 is VH shown in SEQ ID NO: 142 as sequence and VL shown in SEQ ID NO: 166 as sequence; VH shown in SEQ ID NO: 159 as sequence and SEQ ID NO: VH shown in SEQ ID NO: 160 as sequence and VL shown in SEQ ID NO: 168 as sequence; or VH shown in SEQ ID NO: 159 as sequence and VL shown in SEQ ID NO: 169 as sequence;
More preferably, said B7-H4 targeting antigen binding domain comprises a heavy chain as shown in SEQ ID NO: 174 as sequence and a light chain as shown in SEQ ID NO: 198 as sequence; the heavy chain shown in SEQ ID NO: 192 as sequence and the light chain shown in SEQ ID NO: 200 as sequence; or the heavy chain shown in SEQ ID NO: 191 as sequence and SEQ ID NO: as sequence 3. The bispecific antibody of claim 2, comprising a light chain designated 201. said antigen binding domain targeting B7-H4 is in the form of a single VH, tandem VH, ScFv, Fab or IgG; a tandem of 3 or 4 VHs; if in IgG form, the constant region it comprises is preferably derived from human IgG1 containing mutations L234A, L235A and P329G, or human IgG1 containing mutations L234A and L235A The bispecific antibody of claim 2, wherein The 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3, the sequence of HCDR1 is shown in SEQ ID NO: 19 or its variant 1, or SEQ ID NO: 14, and the sequence of HCDR2 is SEQ ID NO: 49 or variant 2 thereof, SEQ ID NO: 51 or SEQ ID NO: 43, wherein the sequence of said HCDR3 is shown in SEQ ID NO: 86 or variant 3 thereof, SEQ ID NO: 96, SEQ ID NO: 89 or SEQ ID NO: 81;
Mutations of said variant 1 comprise one or more of T3I, S6N/R and Y7F; 22;
The mutations of said variant 2 comprise one or more of S1N/D, G2S/A, S3D/G, G5D/F/S/V and S6T/N/D; the sequence of said variant 2 is preferably shown in any of SEQ ID NO: 47-48, SEQ ID NO: 50, SEQ ID NO: 52-58 and SEQ ID NO: 61 in the sequence listing;
The mutations of Mutant 3 are G2R/D/A/K, S3A/T, S4G/N/A/T/H, E5T/V/M/G, T6A, D7G/S, H9Y/S, Y10H , Y11F, N12G/D and V13I/M/T; the amino acid sequence of said variant 3 is preferably SEQ ID NO: 85, SEQ ID NOS: 87-88 and SEQ ID NOS: 90-95 in the sequence listing indicated by any of the
Preferably, said 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 17, 47 and 85, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 48 and 86, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 49 and 87, respectively;
or comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 19, 50 and 88 as sequences, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 20, 51 and 89, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 52 and 90, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 49 and 90, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 21, 53 and 91, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 21, 54 and 92, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 55 and 93, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 49 and 86, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 49 and 94, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 22, 56 and 86, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 57 and 95, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 58 and 96, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 61 and 95, respectively;
or comprising HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 14, 43 and 81, respectively;
More preferably, said 4-1BB-targeted antigen binding domain comprises a heavy chain variable region, one or more of which sequences are shown in SEQ ID NOs: 143-157, 139, 284 or 161;
Even more preferably, according to any one of claims 1-4, wherein said 4-1BB-targeting antigen binding domain comprises a heavy chain shown in SEQ ID NOs: 175-189, 193, 285 or 171 as a sequence. Bispecific antibody. The 4-1BB-targeting antigen binding domain further comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOS: 109, 118 and 128 as sequences, respectively; preferably a light chain variable region shown in sequence SEQ ID NO: 163. more preferably a bispecific antibody according to claim 5, comprising the light chain shown in SEQ ID NO: 195 as sequence. The 4-1BB-targeted antigen-binding domain is in the form of a single VH or tandem VHs, an HCAb form or a ScFv form; 6. A bispecific antibody according to claim 5, which is a tandem of 2, 3 or 4 VHs. said antigen-binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences set forth in SEQ ID NOs: 16, 46 and 84, respectively; and LCDR1, LCDR2 and LCDR3 as sequences shown in SEQ ID NOs: 109, 118 and 128, respectively, and HCDR1, HCDR2 and HCDR3 as sequences shown in SEQ ID NOs: 14, 43 and 81, respectively, wherein said 4-1BB-targeting antigen-binding domain is includes;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and wherein said 4-1BB-targeting antigen binding domain has LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOs: 109, 118 and 128, respectively, and HCDR1, HCDR2 as sequences set forth in SEQ ID NOs: 14, 43 and 81, respectively. and HCDR3;
said antigen-binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 17, 47 and 85, respectively;
said antigen-binding domain targeting B7-H4 comprises LCDR1, LCDR2 and LCDR3 as sequences set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 48 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and the 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 49 and 87, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 19, 50 and 88, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 20, 51 and 89, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and the 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 52 and 90, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 21, 53 and 91, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 21, 54 and 92, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 19, 55 and 93, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 49 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 49 and 94, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 22, 56 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown as sequences in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 as sequences are shown in SEQ ID NOs: 16, 46 and 84, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 57 and 95, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 17, 47 and 85, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 48 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 49 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 19, 49 and 94, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 16, 60 and 84, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 61 and 95, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and wherein said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 48 and 86, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and said 4-1BB-targeted antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 19, 58 and 96, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOs: 18, 57 and 95, respectively;
or LCDR1, LCDR2 and LCDR3, wherein said B7-H4-targeting antigen-binding domain is shown in SEQ ID NOS: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOS: 23, 59 and 98, respectively. and said 4-1BB-targeting antigen binding domain comprises HCDR1, HCDR2 and HCDR3 shown as sequences in SEQ ID NOS: 18, 49 and 90, respectively. Bispecific antibody. said antigen domain targeting B7-H4 is in the form of IgG and said antigen binding domain targeting 4-1BB is in the form of a single VH;
Alternatively, the antigen domain targeting B7-H4 is in the form of IgG and the antigen binding domain targeting 4-1BB is in the form of ScFv;
or, the B7-H4-targeting antigen domain is in the form of IgG, and the 4-1BB-targeting antigen binding domain is in the form of a tandem of two or three VHs;
Alternatively, the B7-H4-targeting antigen domain is in the form of Fab, and the 4-1BB-targeting antigen-binding domain is in the form of HCAb or in the form of HCAb and VH (HCAb-VH form). ;
Alternatively, the antigen domain targeting B7-H4 is in the form of Fab, the antigen binding domain targeting 4-1BB is in the form of VH, and the form of VH is preferably a single VH, two or in the form of a tandem of 3 VHs;
Preferably, said bispecific antibody is of the form:
(1) ScFv is connected to the C-terminus of IgG, VH of said ScFv is connected to said C-terminus; , polypeptide chain 2 is shown in the formula: VHB7-H4-CH1-hinge-CH2-CH3-linker-VH4-1BB-linker-VL4-1BB;
(2) VH or tandem VH is connected to the C-terminus of IgG; shown in VHB7-H4-CH1-hinge-CH2-CH3-linker-(VH4-1BB)n;
(3) Fab is attached to the N-terminus of HCAb; preferably, polypeptide chain 1 of the bispecific antibody of this form is represented by the formula: VHB7-H4-CH1, and polypeptide chain 2 is represented by the formula: VLB7-H4- shown in CL-linker-VH4-1BB-linker-CH2-CH3;
(4) Fab is connected to the N-terminus of the HCAb and VH is connected to the C-terminus of said Fab; or (5) Fab at the N-terminus of the two CH2s of Fc, respectively, and VH or tandem VHs are connected; preferably, the form of the bispecific antibody comprises three polypeptide chains:
Polypeptide chain 1 is represented by the formula: VLB7-H4-CL, polypeptide chain 2 is represented by the formula: VHB7-H4-CH1-hinge-CH2-CH3, and polypeptide chain 3 is represented by the formula: VH4-1BB-linker-CH2. The bispecific antibody of any one of claims 1-8, designated -CH3 or (VH4-1BB)n-linker-CH2-CH3. said linker is selected from the group consisting of SEQ ID NOS: 241-261, 282 and 288-289;
A linker of form (1) preferably comprises the sequence shown in SEQ ID NO:245;
Linkers of form (2) preferably comprise sequences shown in any of SEQ ID NOs:243, 245-247 and SEQ ID NOs:288-289;
A linker of form (3) preferably comprises the sequence shown in SEQ ID NO:250;
A linker of form (4) preferably comprises the sequence shown in SEQ ID NO:282;
10. A bispecific antibody according to claim 9, wherein the linker of form (5) preferably comprises the sequence shown in SEQ ID NO:245. the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 198 and the amino acid sequence of said polypeptide chain 2 is shown in any of SEQ ID NOs: 202-215;
or the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 201 and the amino acid sequence of said polypeptide chain 2 is shown in SEQ ID NOs: 217, 230, 238, 240, 226, 262 or 239;
Alternatively, the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 200, and the amino acid sequence of said polypeptide chain 2 is any of SEQ ID NOS: 218-225, 235-237, 263-268, 274-281, 286 and 287 to show;
Alternatively, the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 201, the amino acid sequence of said polypeptide chain 2 is shown in SEQ ID NO: 227, and the amino acid sequence of said polypeptide chain 3 is shown in SEQ ID NO: 228, 229, 231 or shown at 232;
Alternatively, the duplex of claim 10, wherein the amino acid sequence of said polypeptide chain 1 is shown in SEQ ID NO: 233 and the amino acid sequence of said polypeptide chain 2 is shown in any of SEQ ID NOs: 234, 269-273. specific antibody. An isolated nucleic acid encoding the bispecific antibody of any one of claims 1-11. 13. An expression vector comprising the isolated nucleic acid of claim 12. 14. A host cell, preferably a prokaryotic or eukaryotic cell, comprising an expression vector according to claim 13. 15. A method of making a bispecific antibody, comprising culturing the host cell of claim 14 and obtaining said bispecific antibody from the culture. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-11. The bispecific antibody according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 16 for preventing and/or treating 4-1BB and/or B7-H4 related diseases use in making drugs for
Said disease is preferably cancer, said cancer is preferably breast cancer, ovarian cancer, endometrial cancer, renal cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumor, bile duct cancer, gallbladder cancer , bladder cancer, sarcoma or colorectal cancer; preferably said cancer is breast cancer, ovarian cancer, endometrial cancer, renal cancer or cholangiocarcinoma; more preferably said cancer is breast cancer. A chimeric antigen receptor comprising the bispecific antibody of any one of claims 1-11. An antibody drug conjugate comprising a cytotoxic agent and a bispecific antibody according to any one of claims 1-11; preferably said cytotoxic agent is MMAF or MMAE. The bispecific antibody according to any one of claims 1 to 11, the chimeric antigen receptor according to claim 18, the antibody drug conjugate according to claim 19 and/or the medicament according to claim 16 comprising a composition;
A reagent kit, preferably comprising (i) a device for administering an antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use. A drug kit comprising Kit A and Kit B,
The kit A comprises the bispecific antibody according to any one of claims 1 to 11, the chimeric antigen receptor according to claim 18, the antibody-drug conjugate according to claim 19 and/or claim 16. comprising a pharmaceutical composition according to
Said kit B comprises another anti-tumor antibody or a pharmaceutical composition comprising said other anti-tumor antibody, and/or a hormone preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic agent A drug kit comprising one or more of the group consisting of toxic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines. A method of diagnosing, treating and/or preventing a 4-1BB and/or B7-H4 mediated disease or disorder comprising:
A therapeutically effective amount for a patient in need of the bispecific antibody of any one of claims 1 to 11, the chimeric antigen receptor of claim 18, the antibody-drug conjugate of claim 19, or claim 22. A method comprising administering the pharmaceutical composition of claim 16 or treating a patient in need thereof with the drug kit of claim 21. Said disease or disorder is a tumor, preferably breast cancer, ovarian cancer, endometrial cancer, renal cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumor, bile duct cancer, gallbladder cancer, bladder cancer 23. The method of claim 22, wherein said cancer is cancer, sarcoma or colorectal cancer; preferably said cancer is breast cancer, ovarian cancer, endometrial cancer, renal cancer or cholangiocarcinoma; more preferably said cancer is breast cancer. . A method for immunodetecting or measuring 4-1BB or B7H4, comprising the bispecific antibody according to any one of claims 1 to 11, the chimeric antigen receptor according to claim 18, and the chimeric antigen receptor according to claim 19. 17. A method comprising using an antibody-drug conjugate according to claim 16 or a pharmaceutical composition according to claim 16; preferably said detection is not for diagnostic and/or therapeutic purposes. The bispecific antibody of any one of claims 1 to 11, the chimeric antigen receptor of claim 18, the antibody-drug conjugate of claim 19, or the antibody drug conjugate of claim 16 to a patient in need thereof. and a second therapeutic agent, respectively;

Said second therapeutic agent is preferably another anti-tumor antibody or a pharmaceutical composition comprising said other anti-tumor antibody, and/or a hormone preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent , oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.